Syndecan peptides and polypeptides as inhibitors of cancer

ABSTRACT

The invention provides for peptides from syndecan 4 and methods of use therefor. These peptides can inhibit α6β4 integrin interaction with EGFR, thereby preventing tumor cell growth and tissue invasion.

PRIORITY CLAIM

The present application claims benefit of priority to U.S. ProvisionalApplication Ser. No. 61/669,551 and 61/784,930, filed Jul. 9, 2012 andMar. 14, 2013, respectively, the entire contents of each applicationbeing hereby incorporated by reference.

FEDERAL FUNDING CLAUSE

This invention was made with government support under CA109010 andCA139872 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewithas an ASCII compliant text file named “WARFP0046US_ST25.txt” created onJul. 8, 2013 and having a size of ˜9 KB. The content of theaforementioned file is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to regulation of cell growth, and moreparticularly to regulation of cancer cell growth. In particular,peptides and polypeptides derived from particular regions of thesyndecan 4 molecule has been shown to inhibit engagement of α6β4integrin by EGFR, thereby limiting tissue invasion and tumor cell growthand/or survival.

2. Related Art

It is well established that the EGFR family of receptor tyrosinekinases, in particular EGFR and HER2, have major roles in human cancer(Hynes and Lane, 2005; Hynes and MacDonald, 2009; Scaltriti and Baselga,2006), notably breast cancer, head and neck squamous cell carcinoma(HNSCC), and lung cancer for EGFR. EGFR is a causal agent in triplenegative (ER−, PR−, HER2−) breast cancer (also classified as“basaloid”), which comprises 15-25% of breast cancers (Herschkowitz etal., 2007; Livasy et al., 2006; Perou et al., 2000; Teng et al., 2011).The triple negative (TN) tumors are especially malignant, often strikeyounger women (especially African-Americans) and are resistant totamoxifen therapy (Livasy et al., 2006; Diaz et al., 2005; Haupt et al.,2010; Lu et al., 2008; Carvalho et al., 2010; Friedrichs et al., 1995),the common mode of treatment for most women. These women usually presentas node-positive at first diagnosis and often die of metastatic disease(Dent et al., 2007; Haffty et al., 2006). EGFR has also long beenrecognized to play a central role in progression of squamous cellcarcinoma of the head and neck (H&N), in which it is overexpressed inover 80% of the tumors, and the negative response of H&N patients totherapy (Chang and Califano, 2008; Perez-Ordonez et al., 2006). It iscommonly hyperactivated by overexpression of its ligands (e.g., EGF,TGFα), by activating EGFR mutations, and by its overexpression (Chung etal., 2004; Grandis et al., 1997, Cassell and Grandis, 2010. Furthermore,γ-irradiation, the treatment of choice for many tumors, leads to EGFRactivation by a variety of mechanisms (Zimmerman et al., 2006)—includingincreased EGFR expression (Schmidt-Ullrich et al., 1994), inhibition ofphosphatases that would otherwise maintain receptor quiescence, andpromoting the shedding (e.g., activation) of the membrane-anchoredpro-forms of EGFR ligands (Dent et al., 1999)—leading to significantlypoorer 5-yr survival rates in patients with EGFR-positive tumors.

The α6β4 integrin and its ligand LN332 are upregulated in breast andsquamous cell carcinomas, and are linked to invasion, metastasis andrecurrent disease (Choi and Chen, 2005; Ginos et al., 2004; Patarroyo etal., 2002; Wilhelmsen et al., 2006). Identified as the TSP-180 antigenin mouse tumors (Falcioni et al., 1986), or the A9 antigen in humans(Van Waes et al., 1991; Kimmel and Carey, 1986), high expression of thisantigen predicts a higher rate of early relapse in H&N cancer patients(Wolf et al., 1990; Carey et al., 1987). In more recent studiesexploring its tumor-promoting role using animal models, keratinocytesthat lack expression of the β4 integrin subunit fail to form invasivesquamous cell carcinomas when transformed with ras and IκB, unlike theirnormal counterparts that express the integrin (Dajee et al., 2003; Tranet al., 2008). Despite this seeming importance of the integrin insquamous cell carcinoma, there currently are no therapeutics availableto target its tumor-promoting activities.

A number of labs have investigated the potential role of syndecans inα6β4-mediated cell migration and tumorigenesis. But these studies havefocused largely on syndecans acting as co-receptors with the α6β4integrin in laminin binding rather than a role in directly regulatingα6β4 activation. The phosphorylated and “activated” α6β4 integrinredistributes to the leading edges of invading keratinocytes or tumors;these leading edges overexpress the “unprocessed” form of LN332 thatretains the LG4,5 heparin-binding region that engages syndecans (Amanoet al., 2000; Marinkovich et al., 1992; Matsui et al., 1995; Goldfingeret al., 1999; Goldfinger et al., 1998). Interestingly, recent work fromRouselle's group shows that Sdc1 and Sdc4 bind differently to the LG4,5domain and speculates that this may account for somewhat different cellbehaviors mediated by these two syndecans (Carulli et al., 2012). Otherwork shows that expression of LG4,5 supports tumorigenesis in an animalmodel of squamous cell carcinoma, again suggesting a potential role forsyndecans in tumorigenesis (Tran et al., 2008), although it isadmittedly indirect.

Although not widely appreciated, the α6β4 integrin is also expressed onvascular endothelial cells in vivo, where its function in hemidesmosomesallows the endothelium to resist frictional forces as it does onstratified epithelia. Giancotti has shown a clear role for α6β4 integrinin tumor angiogenesis and that α6β4 is expressed in the vasculature ofseveral tumor types (prostate, breast, glioma, papillary thyroid,melanoma) (Nikolopoulos et al., 2004). Although not studied extensively,it clear that endothelial cells express EGFR family members (Amin etal., 2006) and that tumor endothelial cells upregulate the expression ofEGFR in particular (Amin et al., 2006; Bohling et al., 1996; Bruns etal., 2000; Kedar et al., 2002; Huang et al., 2002). Despite this seemingimportance of the integrin in squamous cell carcinoma and tumor-inducedangiogenesis, there currently are no therapeutics available to targetits tumor-promoting activities.

Work from a variety of laboratories has shown a linkage between the α6β4integrin and EGFR in breast and other cancers (Lu et al., 2008; Folgieroet al., 2008). This integrin in normal cells assembles with laminin inthe basement membrane underlying basal epithelial cells as well asendothelial cells lining blood vessels, forming stable hemidesmosomes inwhich the long (ca. 1000 amino acid) cytoplasmic domain of the β4subunit anchors to the keratin filament network in the cytoplasm of thecell (Wilhelmsen et al., 2006; Hopkinson and Jones, 2000; Nievers etal., 1999). In contrast to this “stabilizing” role, however, theintegrin takes part in the invasion, proliferation and survival oftumors that overexpress the receptor tyrosine kinases HER2, EGFR, orc-Met—leading to the assembly of these kinases with the integrin(Wilhelmsen et al., 2006; Agazie and Hayman, 2003; Mainiero et al.,1996; Mariotti et al., 2001; Bertotti et al., 2005; Bertotti et al.,2006; Bon et al., 2007; Falcioni et al., 1997; Gambaletta et al., 2000;Santoro et al., 2003; Trusolino et al., 2001; Tsuruta et al., 2008;Giancotti, 2007). There is evidence for this in TN breast tumors,especially, as α6β4 integrin and EGFR overexpression are causally linkedin this disease and lead to poor prognosis (Lu et al., 2008). Whencoupled with the integrin, signaling from these kinases disrupts thehemidesmosome (Rabinovitz et al., 2004; Wilhelmsen et al., 2007) andleads to tyrosine phosphorylation of the β4 cytoplasmic domain,providing docking sites for signaling effectors that drive tumor cellproliferation, invasion and survival (Wilhelmsen et al., 2006; Mariottiet al., 2001; Bertotti et al., 2006; Wilhelmsen et al., 2007; Mainieroet al., 1997; Shaw et al., 1997; Guo et al., 2006; Merdek et al., 2007;Dutta and Shaw, 2008; Datta et al., 1999; Dans et al., 2001; Shaw, 2001;Yang et al., 2010). The distal third of the β4 tail containing thesephosphorylation sites has thus been termed the β4 “signaling domain”(Guo et al., 2006) (FIG. 1). In studies using the MMTV-Neu mouse modelof HER2+ breast cancer, replacement of native β4 with a β4 mutant(β4^(1355T)) lacking this signaling domain acts as a suppressor ofbreast cancer (Guo et al., 2006), suggesting that the wild type β4receptor normally couples with HER2 to drive tumorigenesis of HER2+breast cancer as well. Work utilizing a number of mammary carcinoma celllines, focusing mostly on HER2+ cells, also shows that HER2/α6β4signaling is critical for invasion and survival of these tumors(Falcioni et al., 1997; Gambaletta et al., 2000; Guo et al., 2006).Complementing their expression in the tumors, HER2 and EGFR are alsoexpressed in endothelial cells, especially those induced by tumors (Aminet al., 2006; Bruns et al., 2000; Kedar et al., 2002), and couple withthe α6β4 integrin during tumor-induced angiogenesis (Nikolopoulos etal., 2004).

SUMMARY OF THE INVENTION

Thus, in accordance with the present invention there is provided anisolated and purified peptide segment consisting of between 25 and 100amino acid residues and comprising about 45 residues of SEQ ID NO:1including residues 87-131 (SEQ ID NO: 4). The peptide may be 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100amino acid residues in length. The peptide may be between 45 and 54amino acid residues in length, between 31 and 40 amino acid residues, 45and 65 amino acid residues in length or between 45 and 75 amino acidresidues in length. The peptide may consists essentially of or consistof residues 87-131 (SEQ ID NO: 4). The peptide may consist ofessentially of or consist of residues 78-131 (SEQ ID NO: 5). The peptidemay comprise all D amino acid, all L amino acids, or a mixture of D andL amino acids.

Also provided is a nucleic acid encoding a peptide segment consisting ofbetween 45 and 100 amino acid residues and comprising about 45 residuesof SEQ ID NO:1 including residues 87-131 (SEQ ID NO: 4). The encodedpeptide may be 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 65, 70,75, 80, 85, 90, 95 or 100 amino acid residues in length. The encodedpeptide may be between 31 and 40 amino acid residues, 45 and 54 aminoacid residues in length, between 45 and 65 amino acid residues in lengthor between 45 and 75 amino acid residues in length. The encoded peptidemay consists essentially of or consist of residues 87-131 (SEQ ID NO:4). The encoded peptide may consist of essentially of or consist ofresidues 78-131 (SEQ ID NO: 5). The nucleic acid may be in operableconnection to a promoter, and/or located in a replicable vector, such asa viral vector.

In another embodiment, there is provided a method of inhibiting α6β₄integrin interaction with EGFR comprising contacting a EGFR moleculewith a peptide segment consisting of between 45 and 100 amino acidresidues and comprising about 45 residues of SEQ ID NO:1 includingresidues 87-131 (SEQ ID NO: 4). The peptide may be 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acidresidues in length. The peptide may be between 31 and 40 amino acidresidues, between 45 and 54 amino acid residues in length, between 45and 65 amino acid residues in length or between 45 and 75 amino acidresidues in length. The peptide may consists essentially of or consistof residues 87-131 (SEQ ID NO: 4). The peptide may consist ofessentially of or consist of residues 78-131 (SEQ ID NO: 5). The peptidemay comprise all D amino acid, all L amino acids, or a mixture of D andL amino acids. The α6β₄ integrin may be located on the surface of acell, such as a cancer cell, such as a carcinoma, a myeloma, a melanoma,a schwannoma, a malignant peripheral nerve sheath tumor cell or aglioma. The cancer cell may be a metastatic cancer cell or a tumor stemcell. The method may further comprise contacting said cancer cell with asecond cancer inhibitory agent. Contacting may comprise providing tosaid cell an expression construct comprising a nucleic acid encodingsaid peptide segment operably linked to a promoter active in said cell.

Another embodiment encompasses a method of screening for an agent thatinhibits the binding of syndecan-4 and EGFR comprising (a) providing asyndecan-4 or a fragment thereof and a EGFR or a fragment thereof,wherein said syndecan-4 or a fragment thereof and a EGFR or a fragmentthereof are capable of binding each other; (b) contacting the proteinsor fragments of step (a) with a candidate substance; and (c) assessingthe binding of said syndecan-4 or a fragment thereof and said EGFR or afragment thereof, wherein reduced binding in step (c) as compared to thebinding in the absence of said candidate substance identifies saidcandidate substance as an agent that inhibits the binding of syndecan-4and EGFR. The candidate substance may be a protein, a peptide, apeptidometic, a polynucleotide, an oligonucleotide, or a small molecule.One or both of said syndecan-4 or a fragment thereof and/or said EGFR ora fragment thereof may be labeled with a detectable label. Step (c) maycomprise FRET, immunodetection, a gel-shift assay, or a phosphorylationassay. The candidate substance is a peptide segment consistingessentially of or consisting of residues 87-131 (SEQ ID NO: 4) or 78-131(SEQ ID NO: 5). Step (a) may further comprise including α6β₄ or afragment thereof that interacts with syndecan-1 and/or EGFR. The methodmay further comprise a control reaction of assessing the binding of saidsyndecan-4 or a fragment thereof and said EGFR or a fragment thereof inthe absence of said candidate substance. Steps (a)-(c) may be performedin a cell-free system or may be performed in a cell or may be performedin vivo.

In yet a further embodiment, there is provide a method of treating asubject with a cancer, cancer cells of which express α6β₄ integrin andEGFR, comprising contacting said cells with a peptide segment consistingof between 45 and 100 amino acid residues and comprising about 45residues of SEQ ID NO:1 including residues 87-131 (SEQ ID NO: 4). Thepeptide may be 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 65, 70,75, 80, 85, 90, 95 or 100 amino acid residues in length. The peptide maybe between 31 and 40 amino acid residues in length, between 45 and 54amino acid residues in length, between 45 and 65 amino acid residues inlength or between 45 and 75 amino acid residues in length. The peptidemay consists essentially of or consist of residues 87-131 (SEQ ID NO:4). The peptide may consist of essentially of or consist of residues78-131 (SEQ ID NO: 5). The peptide may comprise all D amino acid, all Lamino acids, or a mixture of D and L amino acids. The subject may be anon-human mammal, such as a human. The cancer may be a carcinoma, amyeloma, a melanoma or a glioma. The peptide may be administereddirectly to said cancer cells, local to said cancer cells, regional tosaid cancer cells, or systemically. The method may further compriseadministering to said subject a second cancer therapy selected fromchemotherapy, radiotherapy, immunotherapy, hormonal therapy, or genetherapy. The method may further comprise administering said peptide tosaid subject more than once.

In still another embodiment, there is provided a method of inhibitingscarring in a subject to comprising administering to said subject apeptide segment consisting of between 45 and 100 amino acid residues andcomprising about 45 residues of SEQ ID NO:1 including residues 87-131(SEQ ID NO: 4). The peptide may be 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acid residues inlength. The peptide may be between 45 and 54 amino acid residues inlength, between 45 and 65 amino acid residues in length or between 45and 75 amino acid residues in length. The peptide may consistsessentially of or consist of residues 87-131 (SEQ ID NO: 4). The peptidemay consist of essentially of or consist of residues 78-131 (SEQ ID NO:5). The peptide may comprise all D amino acid, all L amino acids, or amixture of D and L amino acids.

An additional embodiment comprises a method of inhibiting pathologicneovascularization comprising administering to said subject a peptidesegment consisting of between 45 and 100 amino acid residues andcomprising about 45 residues of SEQ ID NO:1 including residues 87-131(SEQ ID NO: 4). The peptide may be 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acid residues inlength. The peptide may be between 45 and 54 amino acid residues inlength, between 45 and 65 amino acid residues in length or between 45and 75 amino acid residues in length. The peptide may consistsessentially of or consist of residues 87-131 (SEQ ID NO: 4). The peptidemay consist of essentially of or consist of residues 78-131 (SEQ ID NO:5). The peptide may comprise all D amino acid, all L amino acids, or amixture of D and L amino acids. The pathologic may neovascularizationinvolves activated vascular endothelial cells.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The word “about” means plus or minus 5% ofthe stated number.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofthe invention that follows.

FIG. 1. Putative model of syndecan-coupling of α6β4 integrin and EGFR incarcinoma and angiogenic endothelial cells. The inventor's findings showthat Sdc4 couples EGFR to the α6β4 integrin. This integrin, togetherwith the α3β1 integrin, is a receptor for LN332 in epithelial cells andendothelial cells. When activated by this kinase, the α6β4 integrinparticipates in matrix adhesion and signaling necessary for cellmigration, but is also critical for cell survival in tumors. EGFR“activates” the α6β4 integrin via its activation of Fyn (Mariotti etal., 2001; Guo et al., 2006; Wang et al., 2010), which phosphorylatesseveral tyrosines in the “β4 signaling domain” (see arrows). Sdc4binding the β4 integrin cytoplasmic domain is critical for thissignaling cascade, ostensibly by bringing the “signaling domain” to themembrane where it can be phosphorylated by Fyn (Mariotti et al., 2001).Mutation of E1729 (E1729A) in the β4 integrin cytoplasmic domaindisrupts Sdc4 binding. This mutant acts as a dominant negative mutant tospecifically disrupt the function of the Sdc4-coupled mechanism duringcell migration and tumor cell survival. An extracellular interactionwith Sdc4 is also critical for the assembly and function of thiscomplex—potentially acting to capture EGFR with the integrin.Recombinant fragments of the Sdc4 ectodomain block the function of theSdc4-coupled EGFR complex. This blocking activity is a putative newsynstatin (SSTN_(EGFR)) that is proposed to inhibit tumor growth,survival and invasion, and tumor-induced angiogenesis.

FIG. 2. Co-immunoprecipitation of α6β4 together with Sdc4 and EGFR fromHNSCC cells. Sdc4, β4 integrin, and EGFR were immunoprecipitated fromlysates of HNSCC cells and probed on Western blots for β4 integrin orfor EGFR (UM-SCC47 cells only). Note that the integrin and EGFRco-precipitate with Sdc4 and with each other. Note that the integrinsubunit is sometimes observed as multiple bands, possibly due toglycosylation differences. (Intervening lanes separating the IgG controland Sdc4 were removed for clarity of presentation).

FIG. 3. Sdc4-coupled EGFR/α6β4 complex in MCF10 and BT474 human mammarycells. Antibodies to Sdc4 or Sdc1 (as a control), β4 integrin, or EGFRwere used to immunoprecipitate the receptors from lysates of MCF10Anormal mammary epithelial cells or BT474 (HER2+) breast carcinoma grownin serum. Blots were then probed for co-precipitation with EGFR.

FIGS. 4A-B. Sdc4 is required for EGFR/α6β4-dependent HaCat keratinocytemigration. (FIG. 4A) HaCat keratinocytes deposit laminin 332 (LN332) andmigrate upon this using α3β1 and α6β4 integrins to close a scratch woundin the monolayer. Migration induced by addition of 10 ng/ml EGF(EGF-mediated chemotaxis) is blocked by antibodies against eitherintegrin, or by BM165 which targets their binding site in LN332. Notethat EGF chemotaxis depends on EGFR, as it is blocked by 1 μM Iressa(also called “gefitinib), which blocks EGFR and not by the tyrphostinAG825 that blocks HER2 kinase expressed by these cells but plays a rolein haptotactic migration by these cells. (FIG. 4B) The potential rolesof Sdc1 (as a control) or Sdc4 in wound closure is tested by silencingtheir expression, together with attempting to rescue with eitherwild-type mouse Sdc1 or Sdc4, or mouse mutants unable to engage the β4cytoplasmic domain (mSdc1ΔC2 or mSdc4ΔC2). EGF-induced chemotaxis isblocked by silencing endogenous Sdc4, and this cannot be rescued bySdc4ΔC2 that fails to engage the α6β4 integrin. Silencing Sdc1 has onlya minor effect, indicating that the EGFR/α6β4 complex is regulated byits interaction with Sdc4.

FIGS. 5A-B. Sdc4-specific binding site in β4 integrin cytoplasmicdomain. (FIG. 5A) Syndecans. The entire cytoplasmic domains of Sdc1 (SEQID NO: 9) and Sdc4 (SEQ ID NO: 10) are shown (Rapraeger & Ott, 1998). C1and C2 regions are conserved across the syndecan family, whereas the Vregion is syndecan-specific. (FIG. 5B) β4 integrin. The last 30 aminoacids of the β4 cytoplasmic domain (over 1,000 amino acids long) areshown (SEQ ID NO: 11), focusing on the last 24 amino acids necessary tobind Sdc1 and Sdc4, as the β4^(Δ1729-1752) truncation mutant fails tobind either syndecan. Mutation of E1729 to alanine (E1729A) specificallydisrupts binding to Sdc4 and the R1733A mutant fails to bind Sdc1.

FIGS. 6A-B. S1ED and S4ED capture HER2 and EGFR, respectively, from celllysates. (FIG. 6A) Mouse syndecan chimeras in which their ectodomainsare switched are expressed in HaCat cells, then immunoprecipitated withantibodies specific for the mouse syndecan ectodomains. Note that EGFRco-precipitate with mSdc1 bearing the mSdc4 ectodomain (precipitated bymS4 antibody), and mSdc4 no longer captures these receptors when itsectodomain is swapped for that of Sdc1. Also note that these complexesassemble only upon EGF treatment. (FIG. 6B) Recombinant Sdc1 and Sdc4ectodomains (GST-S1ED and GST-S4ED) on glutathione beads are incubatedwith lysates of EGF-stimulated A431 carcinoma cells, then analyzed forthe capture of HER2 or EGFR on western blots. HER2 or EGFR areimmunoprecipitated from the lysates directly for comparison. Note thatS1ED captures only HER2 and S4ED captures only EGFR.

FIG. 7. EGFR-dependent migration of MCF10A mammary epithelial cells isblocked by recombinant Sdc4 ectodomain, a peptide representing theactive site in Sdc4 (amino acids 87-131) or a Sdc4-specific dominantnegative α6β4 integrin. Migration of human MCF10A mammary epithelialcells in response to EGF (10 ng/ml) is tested in 12 hr scratch woundclosure assays. Peptide competition: Sdc4 ectodomain (GST-S4ED) fusionprotein blocks EGF-stimulated migration by 80% at 10 μM, whereasGST-S1ED (from Sdc1, used as a control) has no effect. Using this assayto test shorter Sdc4 peptides, the inventor found that a syntheticpeptide comprising amino acids 87-131 in the Sdc4 ectodomain is at leastas effective as the entire ectodomain, displaying an IC₅₀ of 0.1-0.3 μM.Dominant-negative mutants: A β4 cytoplasmic domain mutant (β4^(E1729A))that cannot engage Sdc4 blocks EGF-stimulated chemotaxis, thusdisrupting the activity of the endogenous β4 integrin (dominant negativemutant). A β4 integrin mutant (β4^(R1733A)) that cannot engage Sdc1 butstill binds Sdc4 has no effect (used as a control).

FIGS. 8A-C. Schematic summary of experiments using either recombinantGST fusion proteins or synthetic peptides to disrupt Sdc4-dependentcoupling of EGFR to α6β4 integrin necessary for EGF-stimulated cellmigration. GST-fusion proteins or synthetic peptides were added atconcentrations ranging from 0.1 to 30 μM to the culture medium of humanHaCat keratinocytes or MCF10A mammary epithelial cells in the presenceor absence of 20 ng/ml EGF. The ability of the constructs to inhibit themigration of the cells to close a scratch wound in confluent monolayerswas determined and express as the concentration of inhibitor necessaryto cause 50% inhibition of cell migration. (FIG. 8A) Competition usingrecombinant GST-fusion proteins. GST-Sdc4 fusion proteins bearingtruncations or mutations were assessed for their competitive activity.Proteins that retained the sequence from amino acids 87 to 131 (SEQ IDNO: 4) were found to fully active, tentatively identifying this as theactive site. (FIG. 8B) Schematic representation of the active site inSdc4. The active site is shown as a juxtamembrane site in the ectodomainof Sdc4. TM is the transmembrane domain and CYTO is the cytoplasmicdomain. The sequence of a peptide derived from this site (SSTN_(EGFR))(SEQ ID NO: 4) is shown along with its homology across other mammalianSdc4 species (SEQ ID NO: 12) and its homology to Sdc4 derived fromzebrafish (SEQ ID NO: 13), identifying highly conserved amino acids atthe extreme N- and C-terminus of the peptide. (FIG. 8C) Competition withsynthetic peptides. Peptides were purchased from commercial sources fortesting as a putative synstatin inhibitory peptide (SSTN_(EGFR)).Mutation of isoleucine at position 89 to alanine (189A), or mutation ofamino acids 99-108 to alanine (99-108A) reduce the activity by 10-fold.Truncation of amino acids 126-131 at the C-terminus (Δ126-131) reducesthe activity by at least 100-fold.

FIGS. 9A-F. SSTN treatment of normal (nontransformed) epithelial cellsversus carcinoma (transformed) cells. Cells are plated for one day, thentreated with either 3-30 μM SSTN_(IGF1R) (SSTN-I), 3-30 μM SSTN_(EGFR)(SSTN-E), 3-30 μM SSTN_(HER2) (SSTN-H) or combinations of the threepeptides, each at 10 μM to test their additive effect. Total cell number(a combination of cell proliferation and cell death) is measured usingthe CellTiterGLO assay (Promega) and is plotted as a percentage ofuntreated cells after either 4 or 7 days of treatment (less than 0%demonstrates cell death). The treatment times are chosen to reflect onlymodest effects of the peptides used singly, so that the more pronouncedeffect of peptides used in combination can still be observed. Treatmentfor longer times leads to significant cell death observed even withsingle peptides alone. (FIG. 9A) HaCat human keratinocytes, an exampleof a normal stratified epithelium, (FIG. 9B) UM-SCC47 cells, a squamouscell carcinoma derived from the tongue of a male patient (a stratrifiedepithelium), (FIG. 9C) SCC25 cells, another squamous cell carcinoma fromthe tongue of a male patient), (FIG. 9D) MCF10A cells, a normal humanbreast epithelium, (FIG. 9E) MDA-MB-468 cells, a triple-negative (TN)breast carcinoma, and (FIG. 9F) SKBr3 cells, a HER2+ human breastcarcinoma. Note that all four carcinomas are inhibited by SSTN_(EGFR),and this is enhanced when SSTN_(EGFR) is combined with the other SSTNpeptides, leading to obvious cell death (e.g., the number of cells atthe end of the experiment are less than at the beginning).

FIG. 10. S4ED blocks MDA-MB-468 breast carcinoma growth in vivo. HumanMDA-MB-468 breast carcinoma cells were implanted subcutaneously on theflank of nude (nu/nu) mice and allowed to grow for 2 weeks to formpalpable tumors (dotted blue line indicates size after 2 weeks). At thattime, systemic delivery (0.25 μL/hr) of 400 μM recombinant GST-S4ED, GSTalone or saline alone (no treatment) was initiated by surgicalimplantation of Alzet pumps on the behind the shoulder blades of theanimals for 4 wks, followed by replacement with pumps containing freshprotein for an additional 4 wks. Tumor size was measured with calipersat 2 week intervals. Error bars denote S.D., 10 tumors/treatment group.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

It is increasingly appreciated that growth factor receptors andextracellular matrix receptors work closely together to regulate cellproliferation, invasion and survival, and may do so as macromolecularassemblies at the cell surface. Indeed, as discussed extensively above,EGFR is known to be coupled with the α6β4 integrin and signaling fromthis receptor assembly is implicated in both tumorigenesis andtumor-induced angiogenesis. However, the means by which these receptorsare coupled remains largely unknown.

The inventor has now discovered that the EGFR/α6β4 integrin assembly isregulated by yet another class of receptors—the syndecan family ofmatrix receptors. In this novel mechanism, syndecan-4 (Sdc4) links theα6β4 integrin to EGFR—linkage that is required for tumor cell survival.Importantly, the linkage relies on a highly specific motif in theextracellular domain of Sdc4. This motif, when supplied as a solublepeptide, competes with the signaling from the Sdc4-coupled α6β4integrin/EGFR complex, presumably by disrupting their association withthe syndecan. This inhibitory activity is designated “synstatin-EGFR”(abbreviated SSTN_(EGFR)).

This interaction is illustrated in the model of Sdc4 provided in FIG. 1.All four members of the syndecan family engage the cytoplasmic domain ofthe β4 integrin, but the focus here will be on Sdc4, which is expressedon epithelial cells (Bernfield et al., 1992; David et al., 1992) andco-immunoprecipitates with the α6β4 integrin and EGFR from activatedkeratinocytes, A431 cervical carcinoma cells, breast carcinoma cells andHN squamous carcinoma cells (our preliminary data) (FIGS. 4A-B and TableI). Importantly, EGFR docks only with Sdc4/α6β4 and not with the othersyndecans. The interaction site capturing the EGFR appears to be in theSdc4 extracellular domain, which is distinct from that of othersyndecans, and the inventor finds that recombinant Sdc4 peptidesmimicking these interaction sites (e.g., SSTN_(EGFR)) block thisinteraction. In addition to this extracellular site, the inventor findsthat the syndecans also engage distinct sites in the β4 cytoplasmicdomain. Sdc4 binding its site in the β4 tail is essential for signalingby this complex, as mutation of this Sdc4-specific site generates a β4dominant-negative mutant that specifically blocks the Sdc4-coupledsignaling mechanism.

I. SYNDECANS

A. The Syndecan Family

Cell surface adhesion receptors physically bind cells to theirextracellular matrix (ECM) and couple such interactions to intracellularsignaling mechanisms which influence gene expression, cell morphology,motility, growth, differentiation and survival (Roskelley et al., 1995;Miranti and Brugge, 2002). Many ECM ligands contain closely spacedproteoglycan- and integrin-binding domains, indicating that themolecular mechanisms by which cells recognize and interact with theirextracellular milieu may involve the formation of signaling complexescontaining both proteoglycans and integrins. Consequentially, these twotypes of receptors may act in concert to modulate cell adhesion andmigration. While the role of integrins in cell adhesion and signaling iswell established, the role of heparan sulfate proteoglycans (HSPGs) isnot well characterized.

The vertebrate syndecans are a family of four transmembrane HSPGs.Endowed by their heparan sulfate (HS) chains, syndecans bind a varietyof ECM ligands, including fibronectin (FN), laminin (LN), tenascin,thrombospondin (TSP), vitronectin (VN) and the fibrillar collagens (COL)(Bernfield et al., 1999). While the syndecan HS chains are essential formatrix binding, less is known about the role of syndecan core proteinsin cell adhesion signaling, although the core protein can affect ligandbinding interactions, as well as occupancy induced signaling (Rapraegerand Ott, 1998; Rapraeger, 2000).

The syndecans display a high degree of conservation within their coreproteins both across species and across family members. Like theintegrins, the syndecans lack intrinsic signaling activity. Their shortcytoplasmic tails (ca. 30 aa) consist of three regions, two of which areconserved amongst the four syndecans (C1 and C2) and which flank anintervening variable (V) region. Proteins known to interact with theseconserved domains are believed to link syndecan ligand bindinginteractions to the transduction of intracellular signals (Couchman etal., 2001). Each family member is uniquely defined by its ectodomainsand the V-regions of its cytoplasmic tail. Divergence within theseregions is believed to confer separate and distinct functions to eachindividual family member. Distinct roles for the V-regions of Sdc-2 and-4 in matrix assembly and focal adhesion formation respectively havebeen described (Klass et al., 2000; Woods and Couchman, 2001); however,specific functions for the syndecan ectodomains are almost whollyunknown with the noted exception of Sdc-1 and -4 which contain bindingsites for as yet unidentified cell surface receptor(s) (McFall andRapraeger, 1997; McFall and Rapraeger, 1998).

B. Syndecan Function in Cell Adhesion and Spreading

Current evidence suggests that the syndecan core proteins participate inadhesion-mediated signaling in collaboration with co-receptors at thecell surface. One example is Sdc-4 in focal adhesion and stress fiberformation, which requires both Sdc-4 and integrin engagement whereasneither is sufficient alone (Woods et al., 1986; Izzard et al., 1986;Streeter and Rees, 1987; Singer et al., 1987). The requirement for Sdc-4ligation can be overcome by treatment with phorbol esters (Woods andCouchman, 1994) or lysophosphatidic acid (LPA) (Saoncella et al., 1999)implicating PKC and RhoA in Sdc-4 signaling. While the mechanism bywhich Sdc-4 contributes to RhoA activation is not clear, it is knownthat Sdc-4 interacts directly with PKCα as well as phosphatidyl inositol4,5 bisphosphate (PIP2) via its cytoplasmic tail and these interactionspotentiate PKCα activity (Oh et al., 1997a; Oh et al., 1997b; Oh et al.,1998; Baciu and Goetinck, 1995).

C. Syndecan-4

Syndecan-4 is a protein that in humans is encoded by the SDC4 gene. Thisgene is found on chromosome 20, while a pseudogene has been found onchromosome 22. Syndecan-4 is a transmembrane (type I) heparan sulfateproteoglycan that functions as a receptor in intracellular signaling.The protein is found as a homodimer and is a member of the syndecanproteoglycan family. Syndecan-4 is upregulated in osteoarthritis andinhibition of syndecan-4 reduces cartilage destruction in mouse modelsof OA.

II. Integrins and EGFR

A. α6β4 Integrin

Integrins are receptors that mediate attachment between a cell and thetissues surrounding it, which may be other cells or the ECM. They alsoplay a role in cell signaling and thereby regulate cellular shape,motility, and the cell cycle.

Typically, receptors inform a cell of the molecules in its environmentand the cell responds. Not only do integrins perform this outside-insignalling, but they also operate an inside-out mode. Thus, theytransduce information from the ECM to the cell as well as reveal thestatus of the cell to the outside, allowing rapid and flexible responsesto changes in the environment, for example to allow blood coagulation byplatelets.

There are many types of integrins, and many cells have multiple types ontheir surface. Integrins are of vital importance to all animals and havebeen found in all animals investigated, from sponges to mammals.Integrins have been extensively studied in humans.

Integrins work alongside other proteins such as cadherins,immunoglobulin superfamily cell adhesion molecules, selectins andsyndecans to mediate cell—cell and cell—matrix interaction andcommunication. Integrins bind cell surface and ECM components such asfibronectin, vitronectin, collagen, and laminin.

B. EGFR

The epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans) isthe cell-surface receptor for members of the epidermal growth factorfamily (EGF-family) of extracellular protein ligands. The epidermalgrowth factor receptor is a member of the ErbB family of receptors, asubfamily of four closely related receptor tyrosine kinases: EGFR(ErbB-1), HER2/c-neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4).Mutations affecting EGFR expression or activity could result in cancer.

EGFR (epidermal growth factor receptor) exists on the cell surface andis activated by binding of its specific ligands, including epidermalgrowth factor and transforming growth factor α (TGFα). ErbB2 has noknown direct activating ligand, and may be in an activated stateconstitutively or become active upon heterodimerization with otherfamily members such as EGFR. Upon activation by its growth factorligands, EGFR undergoes a transition from an inactive monomeric form toan active homodimer—although there is some evidence that preformedinactive dimers may also exist before ligand binding. In addition toforming homodimers after ligand binding, EGFR may pair with anothermember of the ErbB receptor family, such as ErbB2/Her2/neu, to create anactivated heterodimer. There is also evidence to suggest that clustersof activated EGFRs form, although it remains unclear whether thisclustering is important for activation itself or occurs subsequent toactivation of individual dimers.

EGFR dimerization stimulates its intrinsic intracellularprotein-tyrosine kinase activity. As a result, autophosphorylation ofseveral tyrosine (Y) residues in the C-terminal domain of EGFR occurs.These include Y992, Y1045, Y1068, Y1148 and Y1173. Thisautophosphorylation elicits downstream activation and signaling byseveral other proteins that associate with the phosphorylated tyrosinesthrough their own phosphotyrosine-binding SH2 domains. These downstreamsignaling proteins initiate several signal transduction cascades,principally the MAPK, Akt and JNK pathways, leading to DNA synthesis andcell proliferation. Such proteins modulate phenotypes such as cellmigration, adhesion, and proliferation. Activation of the receptor isimportant for the innate immune response in human skin. The kinasedomain of EGFR can also cross-phosphorylate tyrosine residues of otherreceptors it is aggregated with, and can itself be activated in thatmanner.

Mutations that lead to EGFR overexpression (known as upregulation) oroveractivity have been associated with a number of cancers, includinglung cancer, anal cancers and glioblastoma multiforme. In this lattercase a more or less specific mutation of EGFR, called EGFRvIII is oftenobserved. Mutations, amplifications or misregulations of EGFR or familymembers are implicated in about 30% of all epithelial cancers.

Mutations involving EGFR could lead to its constant activation, whichcould result in uncontrolled cell division—a predisposition for cancer.Consequently, mutations of EGFR have been identified in several types ofcancer, and it is the target of an expanding class of anticancertherapies.

The identification of EGFR as an oncogene has led to the development ofanticancer therapeutics directed against EGFR, including gefitinib anderlotinib for lung cancer, and cetuximab for colon cancer.

Many therapeutic approaches are aimed at the EGFR. Cetuximab andpanitumumab are examples of monoclonal antibody inhibitors. However theformer is of the IgG1 type, the latter of the IgG2 type; consequences onantibody-dependent cellular cytotoxicity can be quite different. Othermonoclonals in clinical development are zalutumumab, nimotuzumab, andmatuzumab. The monoclonal antibodies block the extracellular ligandbinding domain. With the binding site blocked, signal molecules can nolonger attach there and activate the tyrosine kinase.

Another method is using small molecules to inhibit the EGFR tyrosinekinase, which is on the cytoplasmic side of the receptor. Without kinaseactivity, EGFR is unable to activate itself, which is a prerequisite forbinding of downstream adaptor proteins. Ostensibly by halting thesignaling cascade in cells that rely on this pathway for growth, tumorproliferation and migration is diminished. Gefitinib, erlotinib, andlapatinib (mixed EGFR and ERBB2 inhibitor) are examples of smallmolecule kinase inhibitors.

There are several quantitative methods available that use proteinphosphorylation detection to identify EGFR family inhibitors. New drugssuch as IRESSA and Tarceva directly target the EGFR. Patients have beendivided into EGFR-positive and EGFR-negative, based upon whether atissue test shows a mutation. EGFR-positive patients have shown animpressive 60% response rate, which exceeds the response rate forconventional chemotherapy.

However, many patients develop resistance. Two primary sources ofresistance are the T790M Mutation and MET oncogene. However, as of 2010there was no consensus of an accepted approach to combat resistance norFDA approval of a specific combination. Preclinical results have beenreported for AP26113 which targets the T790M mutation.

Epidermal growth factor receptor has been shown to interact withAndrogen receptor, ARF4, Beta-catenin, Caveolin 1, Caveolin 3, Cbl gene,CBLB, CBLC, CDC25A, CRK, Decorin, Epidermal growth factor, GRB14, Grb2,Janus kinase 2, MUC1, NCK1, NCK2, PKC alpha, PLCG1, PLSCR1, PTPN1,PTPN11, PTPN6, SH2D3A, SH3 KBP1, SHC1, SOS1, Src, STAT1, STAT3, STAT5A,Ubiquitin C, and Wiskott-Aldrich syndrome protein.

III. SYNDECAN PEPTIDES

A. Structure

The present invention contemplates the design, production and use ofvarious syndecan peptides. The structural features of these peptides areas follows. First, the peptides have no more than about 75 consecutiveresidues of a syndecan (78-131 is 54 amino acids, and residues 87-131 is45 residues). Thus, the term “a peptide having no more than 20consecutive residues,” even when including the term “comprising,” cannotbe understood to comprise a greater number of consecutive syndecanresidues. Second, the peptides will contain the motifs responsible forinteraction with EGFR. In general, the peptides will have, at a minimum,40 consecutive residues of the syndecan, including 45 and 54 residues.

The overall length may be 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75,80, 85, 90 or 100 residues. Ranges of peptide length of 45-50 residues,45-54 residues, 45-60 residues, 45-65 residues, 45-70, residues, 45-75residues, 45-80 residues, 45-85 residues, 45-90 residues, and 45-100residues are contemplated. The number of consecutive syndecan residuesmay be 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60.Ranges of consecutive residues of 45-50 residues, 45-54 residues, 45-60residues, 45-65 residues and 45-70 residues, 45-75, residues, 45-80residues or 45-90 residues are contemplated.

Also as mentioned above, peptides modified for in vivo use by theaddition, at the amino- and/or carboxyl-terminal ends, of a blockingagent to facilitate survival of the peptide in vivo are contemplated.This can be useful in those situations in which the peptide termini tendto be degraded by proteases prior to cellular uptake. Such blockingagents can include, without limitation, additional related or unrelatedpeptide sequences that can be attached to the amino and/or carboxylterminal residues of the peptide to be administered. These agents can beadded either chemically during the synthesis of the peptide, or byrecombinant DNA technology by methods familiar in the art.Alternatively, blocking agents such as pyroglutamic acid or othermolecules known in the art can be attached to the amino- and/orcarboxyl-terminal residues.

B. Synthesis

It will be advantageous to produce peptides using the solid-phasesynthetic techniques (Merrifield, 1963). Other peptide synthesistechniques are well known to those of skill in the art (Bodanszky etal., 1976; Peptide Synthesis, 1985; Solid Phase Peptide Synthelia,1984). Appropriate protective groups for use in such syntheses will befound in the above texts, as well as in Protective Groups in OrganicChemistry, 1973. These synthetic methods involve the sequential additionof one or more amino acid residues or suitable protected amino acidresidues to a growing peptide chain. Normally, either the amino orcarboxyl group of the first amino acid residue is protected by asuitable, selectively removable protecting group. A different,selectively removable protecting group is utilized for amino acidscontaining a reactive side group, such as lysine.

Using solid phase synthesis as an example, the protected or derivatizedamino acid is attached to an inert solid support through its unprotectedcarboxyl or amino group. The protecting group of the amino or carboxylgroup is then selectively removed and the next amino acid in thesequence having the complementary (amino or carboxyl) group suitablyprotected is admixed and reacted with the residue already attached tothe solid support. The protecting group of the amino or carboxyl groupis then removed from this newly added amino acid residue, and the nextamino acid (suitably protected) is then added, and so forth. After allthe desired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to provide the final peptide.The peptides of the invention are preferably devoid of benzylated ormethylbenzylated amino acids. Such protecting group moieties may be usedin the course of synthesis, but they are removed before the peptides areused. Additional reactions may be necessary, as described elsewhere, toform intramolecular linkages to restrain conformation.

Aside from the 20 standard amino acids that can be used, there are avast number of “non-standard” amino acids. Two of these can be specifiedby the genetic code, but are rather rare in proteins. Selenocysteine isincorporated into some proteins at a UGA codon, which is normally a stopcodon. Pyrrolysine is used by some methanogenic archaea in enzymes thatthey use to produce methane. It is coded for with the codon UAG.Examples of non-standard amino acids that are not found in proteinsinclude lanthionine, 2-aminoisobutyric acid, dehydroalanine and theneurotransmitter gamma-aminobutyric acid. Non-standard amino acids oftenoccur as intermediates in the metabolic pathways for standard aminoacids—for example ornithine and citrulline occur in the urea cycle, partof amino acid catabolism. Non-standard amino acids are usually formedthrough modifications to standard amino acids. For example, homocysteineis formed through the transsulfuration pathway or by the demethylationof methionine via the intermediate metabolite S-adenosyl methionine,while hydroxyproline is made by a posttranslational modification ofproline.

C. Linkers

Linkers or cross-linking agents may be used to fuse syndecan peptides toother proteinaceous sequences. Bifunctional cross-linking reagents havebeen extensively used for a variety of purposes including preparation ofaffinity matrices, modification and stabilization of diverse structures,identification of ligand and receptor binding sites, and structuralstudies. Homobifunctional reagents that carry two identical functionalgroups proved to be highly efficient in inducing cross-linking betweenidentical and different macromolecules or subunits of a macromolecule,and linking of polypeptide ligands to their specific binding sites.Heterobifunctional reagents contain two different functional groups. Bytaking advantage of the differential reactivities of the two differentfunctional groups, cross-linking can be controlled both selectively andsequentially. The bifunctional cross-linking reagents can be dividedaccording to the specificity of their functional groups, e.g., amino-,sulfhydryl-, guanidino-, indole-, or carboxyl-specific groups. Of these,reagents directed to free amino groups have become especially popularbecause of their commercial availability, ease of synthesis and the mildreaction conditions under which they can be applied. A majority ofheterobifunctional cross-linking reagents contains a primaryamine-reactive group and a thiol-reactive group.

In another example, heterobifunctional cross-linking reagents andmethods of using the cross-linking reagents are described in U.S. Pat.No. 5,889,155, specifically incorporated herein by reference in itsentirety. The cross-linking reagents combine a nucleophilic hydrazideresidue with an electrophilic maleimide residue, allowing coupling inone example, of aldehydes to free thiols. The cross-linking reagent canbe modified to cross-link various functional groups and is thus usefulfor cross-linking polypeptides. In instances where a particular peptidedoes not contain a residue amenable for a given cross-linking reagent inits native sequence, conservative genetic or synthetic amino acidchanges in the primary sequence can be utilized.

Another use of linkers in the context of peptides as therapeutics is theso-called “Stapled Peptide” technology of Aileron Therapeutics. Thegeneral approach for “stapling” a peptide is that two key residueswithin the peptide are modified by attachment of linkers through theamino acid side chains. Once synthesized, the linkers are connectedthrough a catalyst, thereby creating a bridge that physically constrainsthe peptide into its native c′-helical shape. In addition to helpingretain the native structure needed to interact with a target molecule,this conformation also provides stability against peptidases as well ascell-permeating properties. U.S. Pat. Nos. 7,192,713 and 7,183,059,describing this technology, are hereby incorporated by reference. Seealso Schafineister et al., 2000.

D. Design, Variants and Analogs

Having identified structures in EGFR interaction with α6β4 integrins,the inventor also contemplates that variants of the sequences may beemployed. For example, certain non-natural amino acids that satisfy thestructural constraints of the sequences may be substituted without aloss, and perhaps with an improvement in, biological function. Inaddition, the present inventor also contemplates that structurallysimilar compounds may be formulated to mimic the key portions of peptideor polypeptides of the present invention. Such compounds, which may betermed peptidomimetics, may be used in the same manner as the peptidesof the invention and, hence, also are functional equivalents.

Certain mimetics that mimic elements of protein secondary and tertiarystructure are described in Johnson et al. (1993). The underlyingrationale behind the use of peptide mimetics is that the peptidebackbone of proteins exists chiefly to orient amino acid side chains insuch a way as to facilitate molecular interactions, such as those ofantibody and/or antigen. A peptide mimetic is thus designed to permitmolecular interactions similar to the natural molecule.

Methods for generating specific structures have been disclosed in theart. For example, α-helix mimetics are disclosed in U.S. Pat. Nos.5,446,128; 5,710,245; 5,840,833; and 5,859,184. Methods for generatingconformationally restricted β-turns and β-bulges are described, forexample, in U.S. Pat. Nos. 5,440,013; 5,618,914; and 5,670,155. Othertypes of mimetic turns include reverse and γ-turns. Reverse turnmimetics are disclosed in U.S. Pat. Nos. 5,475,085 and 5,929,237, andγ-turn mimetics are described in U.S. Pat. Nos. 5,672,681 and 5,674,976.

As used herein, “molecular modeling” means quantitative and/orqualitative analysis of the structure and function of protein-proteinphysical interaction based on three-dimensional structural informationand protein-protein interaction models. This includes conventionalnumeric-based molecular dynamic and energy minimization models,interactive computer graphic models, modified molecular mechanicsmodels, distance geometry and other structure-based constraint models.Molecular modeling typically is performed using a computer and may befurther optimized using known methods. Computer programs that use X-raycrystallography data are particularly useful for designing suchcompounds. Programs such as RasMol, for example, can be used to generatethree dimensional models. Computer programs such as INSIGHT (Accelrys,Burlington, Mass.), GRASP (Anthony Nicholls, Columbia University), Dock(Molecular Design Institute, University of California at San Francisco),and Auto-Dock (Accelrys) allow for further manipulation and the abilityto introduce new structures. The methods can involve the additional stepof outputting to an output device a model of the 3-D structure of thecompound. In addition, the 3-D data of candidate compounds can becompared to a computer database of, for example, 3-D structures.

Compounds of the invention also may be interactively designed fromstructural information of the compounds described herein using otherstructure-based design/modeling techniques (see, e.g., Jackson, 1997;Jones et al., 1996). Candidate compounds can then be tested in standardassays familiar to those skilled in the art. Exemplary assays aredescribed herein.

The 3-D structure of biological macromolecules (e.g., proteins, nucleicacids, carbohydrates, and lipids) can be determined from data obtainedby a variety of methodologies. These methodologies, which have beenapplied most effectively to the assessment of the 3-D structure ofproteins, include: (a) x-ray crystallography; (b) nuclear magneticresonance (NMR) spectroscopy; (c) analysis of physical distanceconstraints formed between defined sites on a macromolecule, e.g.,intramolecular chemical crosslinks between residues on a protein (e.g.,PCT/US00/14667, the disclosure of which is incorporated herein byreference in its entirety), and (d) molecular modeling methods based ona knowledge of the primary structure of a protein of interest, e.g.,homology modeling techniques, threading algorithms, or ab initiostructure modeling using computer programs such as MONSSTER (Modeling OfNew Structures from Secondary and Tertiary Restraints) (see, e.g.,International Application No. PCT/US99/11913, the disclosure of which isincorporated herein by reference in its entirety). Other molecularmodeling techniques may also be employed in accordance with thisinvention (e.g., Cohen et al., 1990; Navia et al., 1992), thedisclosures of which are incorporated herein by reference in theirentirety). All these methods produce data that are amenable to computeranalysis. Other spectroscopic methods that can also be useful in themethod of the invention, but that do not currently provide atomic levelstructural detail about biomolecules, include circular dichroism andfluorescence and ultraviolet/visible light absorbance spectroscopy. Apreferred method of analysis is x-ray crystallography. Descriptions ofthis procedure and of NMR spectroscopy are provided below.

The present invention may utilize L-configuration amino acids,D-configuration amino acids, or a mixture thereof. While L-amino acidsrepresent the vast majority of amino acids found in proteins, D-aminoacids are found in some proteins produced by exotic sea-dwellingorganisms, such as cone snails. They are also abundant components of thepeptidoglycan cell walls of bacteria. D-serine may act as aneurotransmitter in the brain. The L and D convention for amino acidconfiguration refers not to the optical activity of the amino aciditself, but rather to the optical activity of the isomer ofglyceraldehyde from which that amino acid can theoretically besynthesized (D-glyceraldehyde is dextrorotary; L-glyceraldehyde islevorotary).

One form of an “all-D” peptide is a retro-inverso peptide. Retro-inversomodification of naturally occurring polypeptides involves the syntheticassemblage of amino acids with α-carbon stereochemistry opposite to thatof the corresponding L-amino acids, i.e., D-amino acids in reverse orderwith respect to the native peptide sequence. A retro-inverso analoguethus has reversed termini and reversed direction of peptide bonds (NH—COrather than CO—NH) while approximately maintaining the topology of theside chains as in the native peptide sequence. See U.S. Pat. No.6,261,569, incorporated herein by reference.

X-Ray Crystallography.

X-ray crystallography is based on the diffraction of x-radiation of acharacteristic wavelength by electron clouds surrounding the atomicnuclei in a crystal of a molecule or molecular complex of interest. Thetechnique uses crystals of purified biological macromolecules ormolecular complexes (but these frequently include solvent components,co-factors, substrates, or other ligands) to determine near atomicresolution of the atoms making up the particular biologicalmacromolecule. A prerequisite for solving 3-D structure by x-raycrystallography is a well-ordered crystal that will diffract x-raysstrongly. The method directs a beam of x-rays onto a regular, repeatingarray of many identical molecules so that the x-rays are diffracted fromthe array in a pattern from which the structure of an individualmolecule can be retrieved. Well-ordered crystals of, for example,globular protein molecules are large, spherical or ellipsoidal objectswith irregular surfaces. The crystals contain large channels between theindividual molecules. These channels, which normally occupy more thanone half the volume of the crystal, are filled with disordered solventmolecules, and the protein molecules are in contact with each other atonly a few small regions. This is one reason why structures of proteinsin crystals are generally the same as those of proteins in solution.

Methods of obtaining the proteins of interest are described below. Theformation of crystals is dependent on a number of different parameters,including pH, temperature, the concentration of the biologicalmacromolecule, the nature of the solvent and precipitant, as well as thepresence of added ions or ligands of the protein. Many routinecrystallization experiments may be needed to screen all these parametersfor the combinations that give a crystal suitable for x-ray diffractionanalysis. Crystallization robots can automate and speed up work ofreproducibly setting up a large number of crystallization experiments(see, e.g., U.S. Pat. No. 5,790,421, the disclosure of which isincorporated herein by reference in its entirety).

Polypeptide crystallization occurs in solutions in which the polypeptideconcentration exceeds its solubility maximum (i.e., the polypeptidesolution is supersaturated). Such solutions may be restored toequilibrium by reducing the polypeptide concentration, preferablythrough precipitation of the polypeptide crystals. Often polypeptidesmay be induced to crystallize from supersaturated solutions by addingagents that alter the polypeptide surface charges or perturb theinteraction between the polypeptide and bulk water to promoteassociations that lead to crystallization.

Crystallizations are generally carried out between 4° C. and 20° C.Substances known as “precipitants” are often used to decrease thesolubility of the polypeptide in a concentrated solution by forming anenergetically unfavorable precipitating depleted layer around thepolypeptide molecules (Weber, 1991). In addition to precipitants, othermaterials are sometimes added to the polypeptide crystallizationsolution. These include buffers to adjust the pH of the solution andsalts to reduce the solubility of the polypeptide. Various precipitantsare known in the art and include the following: ethanol, 3-ethyl-2-4pentanediol, and many of the polyglycols, such as polyethylene glycol(PEG). The precipitating solutions can include, for example, 13-24% PEG4000, 5-41% ammonium sulfate, and 1.0-1.5 M sodium chloride, and a pHranging from 5.0-7.5. Other additives can include 0.1 M Hepes, 2-4%butanol, 20-100 mM sodium acetate, 50-70 mM citric acid, 120-130 mMsodium phosphate, 1 mM ethylene diamine tetraacetic acid (EDTA), and 1mM dithiothreitol (DTT). These agents are prepared in buffers and areadded dropwise in various combinations to the crystallization buffer.Proteins to be crystallized can be modified, e.g., by phosphorylation orby using a phosphate mimic (e.g., tungstate, cacodylate, or sulfate).

Commonly used polypeptide crystallization methods include the followingtechniques: batch, hanging drop, seed initiation, and dialysis. In eachof these methods, it is important to promote continued crystallizationafter nucleation by maintaining a supersaturated solution. In the batchmethod, polypeptide is mixed with precipitants to achievesupersaturation, and the vessel is sealed and set aside until crystalsappear. In the dialysis method, polypeptide is retained in a sealeddialysis membrane that is placed into a solution containing precipitant.Equilibration across the membrane increases the polypeptide andprecipitant concentrations, thereby causing the polypeptide to reachsupersaturation levels.

In the preferred hanging drop technique (McPherson, 1976), an initialpolypeptide mixture is created by adding a precipitant to a concentratedpolypeptide solution. The concentrations of the polypeptide andprecipitants are such that in this initial form, the polypeptide doesnot crystallize. A small drop of this mixture is placed on a glass slidethat is inverted and suspended over a reservoir of a second solution.The system is then sealed. Typically, the second solution contains ahigher concentration of precipitant or other dehydrating agent. Thedifference in the precipitant concentrations causes the protein solutionto have a higher vapor pressure than the second solution. Since thesystem containing the two solutions is sealed, an equilibrium isestablished, and water from the polypeptide mixture transfers to thesecond solution. This equilibrium increases the polypeptide andprecipitant concentration in the polypeptide solution. At the criticalconcentration of polypeptide and precipitant, a crystal of thepolypeptide may form.

Another method of crystallization introduces a nucleation site into aconcentrated polypeptide solution. Generally, a concentrated polypeptidesolution is prepared and a seed crystal of the polypeptide is introducedinto this solution. If the concentrations of the polypeptide and anyprecipitants are correct, the seed crystal will provide a nucleationsite around which a larger crystal forms.

Yet another method of crystallization is an electrocrystallizationmethod in which use is made of the dipole moments of proteinmacromolecules that self-align in the Helmholtz layer adjacent to anelectrode (see, e.g., U.S. Pat. No. 5,597,457, the disclosure of whichis incorporated herein by reference in its entirety).

Some proteins may be recalcitrant to crystallization. However, severaltechniques are available to the skilled artisan to inducecrystallization. For example, the removal of flexible polypeptidesegments at the amino or carboxyl terminal end of the protein mayfacilitate production of crystalline protein samples. Removal of suchsegments can be done using molecular biology techniques or treatment ofthe protein with proteases such as trypsin, chymotrypsin, or subtilisin.

In diffraction experiments, a narrow and parallel beam of x-rays istaken from the x-ray source and directed onto the crystal to producediffracted beams. The incident primary beams cause damage to both themacromolecule and solvent molecules. The crystal is, therefore, cooled(e.g., to between −220° C. and −50° C.) to prolong its lifetime. Theprimary beam must strike the crystal from many directions to produce allpossible diffraction spots, so the crystal is rotated in the beam duringthe experiment. The diffracted spots are recorded on a film or by anelectronic detector. Exposed film has to be digitized and quantified ina scanning device, whereas the electronic detectors feed the signalsthey detect directly into a computer. Electronic area detectorssignificantly reduce the time required to collect and measurediffraction data. Each diffraction beam, which is recorded as a spot onfilm or a detector plate, is defined by three properties: the amplitude,which is measured from the intensity of the spot; the wavelength, whichis set by the x-ray source; and the phase, which is lost in x-rayexperiments. All three properties are needed for all of the diffractedbeams in order to determine the positions of the atoms giving rise tothe diffracted beams. One way of determining the phases is calledMultiple Isomorphous Replacement (MIR), which requires the introductionof exogenous x-ray scatterers (e.g., heavy atoms such metal atoms) intothe unit cell of the crystal. For a more detailed description of MIR,see U.S. Pat. No. 6,093,573 (column 15) the disclosure of which isincorporated herein by reference in its entirety.

Atomic coordinates refer to Cartesian coordinates (x, y, and zpositions) derived from mathematical equations involving Fouriersynthesis of data derived from patterns obtained via diffraction of amonochromatic beam of x-rays by the atoms (scattering centers) ofbiological macromolecule of interest in crystal form. Diffraction dataare used to calculate electron density maps of repeating units in thecrystal (unit cell). Electron density maps are used to establish thepositions (atomic coordinates) of individual atoms within a crystal'sunit cell. The absolute values of atomic coordinates convey spatialrelationships between atoms because the absolute values ascribed toatomic coordinates can be changed by rotational and/or translationalmovement along x, y, and/or z axes, together or separately, whilemaintaining the same relative spatial relationships among atoms. Thus, abiological macromolecule (e.g., a protein) whose set of absolute atomiccoordinate values can be rotationally or translationally adjusted tocoincide with a set of prior determined values from an analysis ofanother sample is considered to have the same atomic coordinates asthose obtained from the other sample.

Further details on x-ray crystallography can be obtained from co-pendingU.S. Application No. 2005/0015232, U.S. Pat. No. 6,093,573 andInternational Application Nos. PCT/US99/18441, PCT/US99/11913, andPCT/US00/03745. The disclosures of all these patent documents areincorporated herein by reference in their entirety.

NMR Spectroscopy.

Whereas x-ray crystallography requires single crystals of amacromolecule of interest, NMR measurements are carried out in solutionunder near physiological conditions. However, NMR-derived structures arenot as detailed as crystal-derived structures.

While the use of NMR spectroscopy was until relatively recently limitedto the elucidation of the 3-D structure of relatively small molecules(e.g., proteins of 100-150 amino acid residues), recent advancesincluding isotopic labeling of the molecule of interest and transverserelaxation-optimized spectroscopy (TROSY) have allowed the methodologyto be extended to the analysis of much larger molecules, e.g., proteinswith a molecular weight of 110 kDa (Wider, 2000).

NMR uses radio-frequency radiation to examine the environment ofmagnetic atomic nuclei in a homogeneous magnetic field pulsed with aspecific radio frequency. The pulses perturb the nuclear magnetizationof those atoms with nuclei of nonzero spin. Transient time domainsignals are detected as the system returns to equilibrium. Fouriertransformation of the transient signal into a frequency domain yields aone-dimensional NMR spectrum. Peaks in these spectra represent chemicalshifts of the various active nuclei. The chemical shift of an atom isdetermined by its local electronic environment. Two-dimensional NMRexperiments can provide information about the proximity of various atomsin the structure and in three dimensional space. Protein structures canbe determined by performing a number of two- (and sometimes 3- or 4-)dimensional NMR experiments and using the resulting information asconstraints in a series of protein folding simulations.

More information on NMR spectroscopy including detailed descriptions ofhow raw data obtained from an NMR experiment can be used to determinethe 3-D structure of a macromolecule can be found in: Protein NMRSpectroscopy, Principles and Practice, (1996); Gronenborn et al. (1990);and Wider (2000), supra., the disclosures of all of which areincorporated herein by reference in their entirety

Also of interest are peptidomimetic compounds that are designed basedupon the amino acid sequences of compounds of the invention that arepeptides. Peptidomimetic compounds are synthetic compounds having athree-dimensional conformation “motif” that is substantially the same asthe three-dimensional conformation of a selected peptide. The peptidemotif provides the peptidomimetic compound with the ability to inhibitthe interaction of α6β4 and EGFR. Peptidomimetic compounds can haveadditional characteristics that enhance their in vivo utility, such asincreased cell permeability and prolonged biological half-life. Thepeptidomimetics typically have a backbone that is partially orcompletely non-peptide, but with side groups that are identical to theside groups of the amino acid residues that occur in the peptide onwhich the peptidomimetic is based. Several types of chemical bonds,e.g., ester, thioester, thioamide, retroamide, reduced carbonyl,dimethylene and ketomethylene bonds, are known in the art to begenerally useful substitutes for peptide bonds in the construction ofprotease-resistant peptidomimetics.

IV. THERAPIES

A. Pharmaceutical Formulations and Routes of Administration

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions in a form appropriate for theintended application. Generally, this will entail preparing compositionsthat are essentially free of pyrogens, as well as other impurities thatcould be harmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the vector to cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. Such routes includeoral, nasal, buccal, rectal, vaginal or topical route. Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal, or intravenous injection. Suchcompositions would normally be administered as pharmaceuticallyacceptable compositions, described supra. Of particular interest isdirect intratumoral administration, perfusion of a tumor, oradmininstration local or regional to a tumor, for example, in the localor regional vasculature or lymphatic system, or in a resected tumor bed.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional medium or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

For oral administration the polypeptides of the present invention may beincorporated with excipients and used in the form of non-ingestiblemouthwashes and dentifrices. A mouthwash may be prepared incorporatingthe active ingredient in the required amount in an appropriate solvent,such as a sodium borate solution (Dobell's Solution). Alternatively, theactive ingredient may be incorporated into an antiseptic wash containingsodium borate, glycerin and potassium bicarbonate. The active ingredientmay also be dispersed in dentifrices, including: gels, pastes, powdersand slurries. The active ingredient may be added in a therapeuticallyeffective amount to a paste dentifrice that may include water, binders,abrasives, flavoring agents, foaming agents, and humectants.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences,” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

B. Cancer Types and Subjects

Cancer cells to which the methods of the present invention can beapplied include generally any cell that expresses α6β4 integrin, andmore particularly, that overexpresses α6β4 integrin. An appropriatecancer cell can be a breast cancer, lung cancer, colon cancer,pancreatic cancer, renal cancer, stomach cancer, liver cancer, bonecancer, hematological cancer (e.g., leukemia or lymphoma), neural tissuecancer, melanoma, ovarian cancer, testicular cancer, prostate cancer,cervical cancer, vaginal cancer, or bladder cancer cell. In addition,the methods of the invention can be applied to a wide range of species,e.g., humans, non-human primates (e.g., monkeys, baboons, orchimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits,guinea pigs, gerbils, hamsters, rats, and mice.

C. Treatment Methods

Peptides or analogs that inhibit α6β4 integrin engagement of EGFR aregenerally useful as anti-cancer therapeutics or prophylactics. They canbe administered to mammalian subjects (e.g., human breast cancerpatients) alone or in conjunction with other drugs and/or radiotherapy.The compounds can also be administered to subjects that are geneticallyand/or environmentally (due to, for example, physiological and/orenvironmental factors) susceptible to cancer, e.g., subjects with afamily history of cancer (e.g., breast cancer), subjects with chronicinflammation or subject to chronic stress, or subjects that are exposedto natural or non-natural environmental carcinogenic conditions (e.g.,excessive exposure to sunlight, industrial carcinogens, or tobaccosmoke).

When the methods are applied to subjects with cancer, prior toadministration of a compound, the cancer can optionally be tested forα6β4 integrin or EGFR expression or overexpression by methods known inthe art. In this way, subjects can be identified as being susceptible totreatments according to the present invention. Such methods can beperformed in vitro on cancer cells obtained from a subject.Alternatively, in vivo imaging techniques using, for example,radiolabeled antibodies specific for α6β4 integrin or EGFR can beperformed.

The dosage required depends on the choice of the route ofadministration; the nature of the formulation; the nature of thepatient's illness; the subject's size, weight, surface area, age, andsex; other drugs being administered; and the judgment of the attendingphysician. Suitable dosages are in the range of 0.0001 mg/kg-100 mg/kg.Wide variations in the needed dosage are to be expected in view of thevariety of compounds available and the differing efficiencies of variousroutes of administration. For example, oral administration would beexpected to require higher dosages than administration by intravenousinjection. Variations in these dosage levels can be adjusted usingstandard empirical routines for optimization as is well understood inthe art. Administrations can be single or multiple (e.g., 2-, 3-, 4-,5-, 6-, 8-, 10-, 20-, 50-,100-, 150-, or more times). Encapsulation ofthe polypeptide in a suitable delivery vehicle (e.g., polymericmicroparticles or implantable devices) may increase the efficiency ofdelivery, particularly for oral delivery.

D. Scarring and other Pathologic Wound Healing

Wound healing is an essential process in maintaining health. However, incertain instances, wound healing can create health problems. Theseinclude hypertrophic scarring, keloid or dermoid formation, andexuberant granulation. These conditions are often supported bypathologic angiogenesis (discussed below). The present invention may beapplied to address these conditions.

1. Keloids

A keloid is a type of scar which, depending on its maturity, is composedmainly of either type III (early) or type I (late) collagen. It is aresult of an overgrowth of granulation tissue (collagen type 3) at thesite of a healed skin injury which is then slowly replaced by collagentype 1. Keloids are firm, rubbery lesions or shiny, fibrous nodules, andcan vary from pink to flesh-coloured or red to dark brown in colour. Akeloid scar is benign, non-contagious, but sometimes accompanied bysevere itchiness and pain, and changes in texture. In severe cases, itcan affect movement of skin.

Keloids should not be confused with hypertrophic scars, which are raisedscars that do not grow beyond the boundaries of the original wound.Keloids expand in claw-like growths over normal skin. They have thecapability to hurt with a needle-like pain or to itch without warning,although the degree of sensation varies from patient to patient.

If the keloid becomes infected, it may ulcerate. Removing the scar isone treatment option; however, it may result in more severeconsequences: the probability that the resulting surgery scar will alsobecome a keloid is high, usually greater than 50%. Laser treatment hasalso been used with varying degrees of success.

Keloids form within scar tissue. Collagen, used in wound repair, tendsto overgrow in this area, sometimes producing a lump many times largerthan that of the original scar. Although they usually occur at the siteof an injury, keloids can also arise spontaneously. They can occur atthe site of a piercing and even from something as simple as a pimple orscratch. They can occur as a result of severe acne or chickenpoxscarring, infection at a wound site, repeated trauma to an area,excessive skin tension during wound closure or a foreign body in awound. Keloids can sometimes be sensitive to chlorine. Keloid scars cangrow, if they appear at a younger age, because the body is stillgrowing.

Histologically, keloids are fibrotic tumors characterized by acollection of atypical fibroblasts with excessive deposition ofextracellular matrix components, especially collagen, fibronectin,elastin, and proteoglycans. Generally, keloids contain relativelyacellular centers and thick, abundant collagen bundles that form nodulesin the deep dermal portion of the lesion. Keloids present a therapeuticchallenge that must be addressed, as these lesions can cause significantpain, pruritus (itching), and physical disfigurement. They may notimprove in appearance over time and can limit mobility if located over ajoint.

Keloids affect both sexes equally, although the incidence in youngfemale patients has been reported to be higher than in young males,probably reflecting the greater frequency of earlobe piercing amongwomen. There is a fifteen times higher frequency of occurrence in highlypigmented people. Persons of African descent are at increased risk ofkeloid occurrences.

The best treatment is prevention in patients with a knownpredisposition. This includes preventing unnecessary trauma or surgery(including ear piercing, elective mole removal), whenever possible. Anyskin problems in predisposed individuals (e.g., acne, infections) shouldbe treated as early as possible to minimize areas of inflammation.

Intra-Lesional Corticosteroids.

Intra-lesional corticosteroids are first-line therapy for most keloids.A systematic review found that up to 70 percent of patients respond tointra-lesional corticosteroid injection with flattening of keloids,although the recurrence rate is high in some studies (up to 50 percentat five years). While corticosteroids are one of the more commontreatments, injections into and in close proximity to keloid tissue canbe highly painful and can produce undesirable results in femalepatients, as per any other testosterone-based treatment.

Excision.

Scalpel excision may be indicated if injection therapy alone isunsuccessful or unlikely to result in significant improvement. Excisionshould be combined with preoperative, intraoperative, or postoperativetriamcinolone or interferon injections. Recurrence rates from 45 to 100percent have been reported in patients treated with excision alone; thisfalls to below 50 percent in patients treated with combination therapy.

Gel Sheeting.

Both hydrogel and silicone gel sheeting have been used for the treatmentof symptoms (e.g., pain and itching) in patients with establishedkeloids as well as for the management of evolving keloids and theprevention of keloids at the sites of new injuries. While the precisemechanism of action is still poorly understood, there is evidence thatapplication of gel sheeting may reduce the incidence of abnormalscarring. A controlled study found significant changes in growth factorlevels of fibronectin and IL-8 with application of hydrogel sheetingwith respect to normal skin. Silicone sheeting was associated withchanging growth factor levels of only fibronectin.

Cryosurgery.

Most useful in combination with other treatments for keloids. The majorside effect is permanent hypopigmentation, which limits its use inpeople with darker skin.

Radiation Therapy.

Most studies, but not all, have found radiation therapy to be highlyeffective in reducing keloid recurrence, with improvement rates of 70 to90 percent when administered after surgical excision. A small randomizedtrial of treatments after surgery found recurrences in two of sixteenearlobe keloids (13 percent) treated with radiation therapy and in fourof twelve earlobe keloids (33 percent) treated with steroid injections.However, concern regarding the potential long-term risks (e.g.,malignancy) associated with using radiation for an essentially benigndisorder limits its utility in most patients. Only a few cases ofmalignancy that may have been associated with radiation therapy forkeloids have been reported. Although causation cannot be confirmed inthese cases, caution should still be used when prescribing radiationtherapy for keloids, particularly when treating younger patients.Radiation therapy may occasionally be appropriate as treatment forkeloids that are resistant to other therapies. In addition, radiationtherapy may be indicated for lesions that are not amenable to resection.

Interferon Alpha.

Interferon alpha injections may reduce recurrence rates postoperatively.However, all currently available studies of interferon therapy sufferfrom methodologic problems, making an evidence-based recommendationregarding its use difficult.

Pulsed Dye Laser.

Pulsed dye laser treatment can be beneficial for keloids, and appears toinduce keloid regression through suppression of keloid fibroblastproliferation, and induction of apoptosis and enzyme activity.Combination treatment with pulsed dye laser plus intralesional therapywith corticosteroids and/or fluorouracil may be superior to eitherapproach alone.

2. Hypertrophic Scarring

Hypertrophic scars are a cutaneous condition characterized by depositsof excessive amounts of collagen which gives rise to a raised scar, butnot to the degree observed with keloids. Like keloids, they form mostoften at the sites of pimples, body piercings, cuts and burns. Theyoften contain nerves and blood vessels. They generally develop afterthermal or traumatic injury that involves the deep layers of the dermisand express high levels of TGF-β.

When a normal wound heals the body produces new collagen fibers at arate which balances the breakdown of old collagen. Hypertrophic scarsare red and thick and may be itchy or painful. They do not extend beyondthe boundary of the original wound but may continue to thicken for up to6 months. They usually improve over the one or two years but may causedistress due to their appearance or the intensity of the itching, alsorestricting movement if they are located close to a joint.

Hypertrophic scars are more common in the young and people with darkerskin. Some people have an inherited tendency to this type of scarring.It is not possible to completely prevent hypertrophic scars, so anyonewho has suffered one should inform their doctor or surgeon if they needto have surgery. Scar Therapies are available which may speed up theprocess of change from a hypertrophic scar to a flatter, paler one.Scars do not occur in younger people as often as older people becausetheir skin cells replicate more quickly and fill in the wound withnormal skin tissue.

3. Proud Flesh

Granulation tissue is the perfused, fibrous connective tissue thatreplaces a fibrin clot in healing wounds. Granulation tissue typicallygrows from the base of a wound and is able to fill wounds of almost anysize it heals. In addition, it is also found in ulcers like esophagealulcer. However, when the granulation becomes uncontrolled, oftenresulting from improper wound care, a condition known as exuberantgranulation or “proud flesh” results. The scar tissue, if untreated, maycompletely overtake the wound area. Caught early, the condition can betreated by topical or injected steroids, but more advanced cases requiresurgical intervention. Horses are subject to this disease, particularlyin the legs. Also, some individuals of African decent have a geneticpredisposition to exuberant scarring.

E. Pathologic Angiogenesis

Despite the abundancy of angiogenic factors present in differenttissues, endothelial cell turnover in a healthy adult organism isremarkably low with a turnover in the order of thousands of days. Themaintenance of endothelial quiescence is thought to be due to thepresence of endogenous negative regulators. Moreover, positive andnegative regulators often co-exist in tissues with extensiveangiogenesis. These observations have led to the hypothesis thatactivation of the endothelium depends on a balance between theseopposing regulators. If positive angiogenic factors dominate, theendothelium will be activated. Thus, the angiogenic process can bedivided in an activation phase (initiation and progression of theangiogenic process) and a phase of resolution (termination andstabilization of the vessels). It is not yet clear whether theresolution phase is due to upregulation of endogenous inhibitors orexhaustion of positive regulators.

With respect to activated endothelium, an important distinction must bemade between physiological and pathological settings. Although manypositive and negative regulators operate in both, endothelial cellproliferation is tightly controlled in the former, whereas in thelatter, the uncontrolled growth of microvessels may lead to several“angiogenic diseases” in different tissues, such as hemangiomas,psoriasis, Kaposi's sarcoma, ocular neovascularization, rheumatoidarthritis, endometriosis, atherosclerosis, tumor growth and metastasis,myocardial ischemia, peripheral ischemia, cerebral ischemia, woundhealing, reconstructive surgery, and ulcer healing, and these may alsobe advantageously treated with the compositions of the presentinvention. Some of these are discussed in greater detail below.

Hemangiomas are angiogenic diseases, characterized by the proliferationof capillary endothelium with accumulation of mast cells, fibroblastsand macrophages. They represent the most frequent tumors of infancy,occurring more frequently in females than males (3:1 ratio). Hemangiomasare characterized by rapid neonatal growth (proliferating phase). By theage of 6 to 10 months, the hemangioma's growth rate becomes proportionalto the growth rate of the child, followed by a very slow regression forthe next 5 to 8 years (involuting phase). Most hemangiomas occur assingle tumors whereas about 20% of the affected infants have multipletumors, which may appear at any body site. Approximately 5% producelife-, sight-, or limb-threatening complications, with high mortalityrates. The pathogenesis of hemangiomas has not yet been elucidated.However, several immunohistochemical studies have provided insight intothe histopathology of these lesions. In particular, proliferatinghemangiomas express high levels of proliferating cell nuclear antigen(PCNA, a marker for cells in the S phase), type IV collagenase, VEGF andFGF-2. During the involuting phase of hemangiomas, expression of theseangiogenic factors decreases. Furthermore, urinary levels of FGF-2 areelevated during the proliferating phase of hemangioma, but become normalduring involution or after therapy with IFN-α.

Other proliferative disorders of the skin include psoriasis and Kaposi'ssarcoma. Hypervascular psoriatic lesions express high levels of theangiogenic inducer IL-8, whereas the expression of the endogenousinhibitor TSP-1 is decreased. Kaposi's sarcoma (KS) is the most commontumor associated with human immunodeficiency virus (HIV) infection andis in this setting almost always associated with human herpes virus 8(HHV-8) infection. Typical features of KS are proliferatingspindle-shaped cells, considered to be the tumor cells and endothelialcells forming blood vessels. KS is a cytokine-mediated disease, highlyresponsive to different inflammatory mediators like IL-1β, TNF-α andIFN-γ and angiogenic factors. In particular, FGF-2 was found tosynergize with HIV-tat to promote angiogenesis and KS development.Finally, growth of KS, both in vitro and in vivo, could be blocked by anantisense oligonucleotide targeting FGF-2.

Diabetic retinopathy is the leading cause of blindness in the workingpopulation, but ocular neovascularization can also occur upon exposureof preterm babies to oxygen. It is assumed that both forms are inducedby hypoxia in the retina. Elevated levels of the hypoxia-inducibleangiogenic factor VEGF were detected in the aqueous and vitreous of eyeswith proliferative retinopathy.

Excessive production of angiogenic factors from infiltratingmacrophages, immune cells or inflammatory cells may also trigger theformation of pannus, an extensively vascularized tissue that invades anddestroys the cartilage, as seen in rheumatoid arthritis. Moreover,uncontrolled angiogenesis may underlie various female reproductivedisorders, such as prolonged menstrual bleeding or infertility, andexcessive endothelial cell proliferation has been observed in theendometrium of women with endometriosis.

Angiogenesis also contributes to atherosclerosis, a major cause of deathof Western populations. Atherosclerosis is the main cause of heartattack. The walls of the coronary artery are normally free ofmicrovessels except in the atherosclerotic plaques, where there aredense networks of capillaries, known as the vasa vasorum. These fragilemicrovessels can cause hemorrhages, leading to blood clotting, with asubsequent decreased blood flow to the heart muscle and heart attack.Finally, angiogenesis is thought to be indispensable for solid tumorgrowth and metastasis.

V. COMBINATION THERAPIES

Tumor cell resistance to DNA damaging agents represents a major problemin clinical oncology. One goal of current cancer research is to findways to improve the efficacy of chemo- and radiotherapy. One way is bycombining such traditional therapies with gene therapy. In the contextof the present invention, it is contemplated that syndecan peptidetherapy could be used similarly in conjunction with chemotherapeutic,radiotherapeutic, or immunotherapuetic intervention.

To kill cells, inhibit cell growth, inhibit metastasis, inhibitangiogenesis or otherwise reverse or reduce the malignant phenotype oftumor cells, using the methods and compositions of the presentinvention, one would generally contact a target cell with a syndecanpeptide and at least one other therapy. These therapies would beprovided in a combined amount effective to kill or inhibit proliferationof the cell. This process may involve contacting the cells with theagents/therapies at the same time. This may be achieved by contactingthe cell with a single composition or pharmacological formulation thatincludes both agents, or by contacting the cell with two distinctcompositions or formulations, at the same time, wherein one compositionincludes the syndecan peptide and the other includes the agent.

Alternatively, the syndecan treatment may precede or follow the othertreatment by intervals ranging from minutes to weeks. In embodimentswhere the other treatment and the syndecan peptide are appliedseparately to the cell, one would generally ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the therapies would still be able to exert an advantageouslycombined effect on the cell. In such instances, it is contemplated thatone would contact the cell with both modalities within about 12-24 hoursof each other, within about 6-12 hours of each other, or with a delaytime of only about 12 hours. In some situations, it may be desirable toextend the time period for treatment significantly; however, whereseveral days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7or 8) pass between the respective administrations.

It also is conceivable that more than one administration of either thesyndecan peptide or the other therapy will be desired. Variouscombinations may be employed, where the syndecan peptide is “A,” and theother therapy is “B,” as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/BOther combinations are contemplated. Again, to achieve cell killing,both therapies are delivered to a cell in a combined amount effective tokill the cell.

Agents or factors suitable for use in a combined therapy include anychemical compound or treatment method that induces DNA damage whenapplied to a cell. Such agents and factors include radiation and wavesthat induce DNA damage such as, γ-irradiation, X-rays, UV-irradiation,microwaves, electronic emissions, and the like. A variety of chemicalcompounds, also described as “chemotherapeutic” or “genotoxic agents,”are intended to be of use in the combined treatment methods disclosedherein. In treating cancer according to the invention, one would contactthe tumor cells with an agent in addition to the expression construct.This may be achieved by irradiating the localized tumor site withradiation such as X-rays, UV-light, γ-rays or even microwaves.Alternatively, the tumor cells may be contacted with the agent byadministering to the subject a therapeutically effective amount of apharmaceutical composition.

Various classes of chemotherapeutic agents are comtemplated for use incombination with peptides of the present invention. For example,selective estrogen receptor antagonists (“SERMs”), such as Tamoxifen,4-hydroxy Tamoxifen (Afimoxfene), Falsodex, Raloxifene, Bazedoxifene,Clomifene, Femarelle, Lasofoxifene, Ormeloxifene, and Toremifene.

Chemotherapeutic agents contemplated to be of use, include, e.g.,camptothecin, actinomycin-D, mitomycin C. The invention also encompassesthe use of a combination of one or more DNA damaging agents, whetherradiation-based or actual compounds, such as the use of X-rays withcisplatin or the use of cisplatin with etoposide. The agent may beprepared and used as a combined therapeutic composition, or kit, bycombining it with peptides, as described above.

Agents that directly cross-link DNA or form adducts are also envisaged.Agents such as cisplatin, and other DNA alkylating agents may be used.Cisplatin has been widely used to treat cancer, with efficacious dosesused in clinical applications of 20 mg/m² for 5 days every three weeksfor a total of three courses. Cisplatin is not absorbed orally and musttherefore be delivered via injection intravenously, subcutaneously,intratumorally or intraperitoneally.

Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include Adriamycin, also known as Doxorubicin, Etoposide,Verapamil, Podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m² at 21 day intervals for Doxorubicin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally. Microtubuleinhibitors, such as taxanes, also are contemplated. These molecules arediterpenes produced by the plants of the genus Taxus, and includepaclitaxel and docetaxel.

mTOR, the mammalian target of rapamycin, also known as FK506-bindingprotein 12-rapamycin associated protein 1 (FRAP1) is a serine/threonineprotein kinase that regulates cell growth, cell proliferation, cellmotility, cell survival, protein synthesis, and transcription. Rapamycinand analogs thereof (“rapalogs”) are therefore contemplated for use incombination cancer therapy in accordance with the present invention.

Another possible combination therapy with the peptides claimed herein isTNF-α (tumor necrosis factor-alpha), a cytokine involved in systemicinflammation and a member of a group of cytokines that stimulate theacute phase reaction. The primary role of TNF is in the regulation ofimmune cells. TNF is also able to induce apoptotic cell death, to induceinflammation, and to inhibit tumorigenesis and viral replication.

Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage. As such a number ofnucleic acid precursors have been developed. Particularly useful areagents that have undergone extensive testing and are readily available.As such, agents such as 5-fluorouracil (5-FU), are preferentially usedby neoplastic tissue, making this agent particularly useful fortargeting to neoplastic cells. Although quite toxic, 5-FU, is applicablein a wide range of carriers, including topical, however intravenousadministration with doses ranging from 3 to 15 mg/kg/day being commonlyused.

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, x-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageDNA, on the precursors of DNA, the replication and repair of DNA, andthe assembly and maintenance of chromosomes. Dosage ranges for x-raysrange from daily doses of 50 to 200 roentgens for prolonged periods oftime (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosageranges for radioisotopes vary widely, and depend on the half-life of theisotope, the strength and type of radiation emitted, and the uptake bythe neoplastic cells.

The skilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624-652. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

The inventor proposes that the local or regional delivery of syndecanpeptides to patients with cancer will be a very efficient method fortreating the clinical disease. Similarly, the chemo- or radiotherapy maybe directed to a particular, affected region of the subject's body.Alternatively, regional or systemic delivery of expression constructand/or the agent may be appropriate in certain circumstances, forexample, where extensive metastasis has occurred.

Combination with immunotherapy, hormone therapy, toxin therapy andsurgery also is contemplated. In particular, one may employ targetedtherapies such as Avastin, Erbitux, Gleevec, Herceptin and Rituxan.

It also should be pointed out that any of the foregoing therapies mayprove useful by themselves in treating cancer.

VI. EXAMPLES

The following examples are included to demonstrate particularembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constituteparticular modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Materials and Methods

Antibodies and Reagents.

Antibodies used were mouse mAb 3E1 and P1B5 (Chemicon, Temecula, Calif.)to β4 and β1 integrin extracellular domains, respectively; rabbitpolyclonal antibody Ab1922 (Millipore, Billerica, Mass.) to the β4cytoplasmic domain; rabbit antibody BM165 (kindly provided by Dr. PeterMarinkovich, Stanford University, CA) against laminin α3 chain. Theinventors used mouse mAbs B-A38 (Accurate Chemical and Scientific,Westbury, N.Y.) and 150.9 (University of Alabama Hybridoma Facility) tohuman Sdc1 and human Sdc4, respectively and rat mAb 281.2 (65) or KY 8.2(66) against mouse Sdc1 or mouse Sdc4, respectively. HER2 was recognizedby anti-c-ErbB-2 Ab-15 (clone 3B5) (Fisher). EGFR-specific antibody(sc-03-G) was from Santa Cruz.

Dulbecco's modified Eagles medium (DMEM) and rhodamine-conjugatedphalloidin were from Invitrogen (Grand Island, N.Y.);Glutathione-conjugated Sepharose beads were from GE HealthcareBiosciences Corp (Piscataway, N.J.); Human recombinant epidermal growthfactor (rhEGF) was from Sigma Aldrich (St. Louis, Mo.); ErbB2 inhibitor(AG825) was from Chemicon; EGFR inhibitor (Iressa) was kindly providedby Dr. Deric Wheeler (University of Wisconsin, WI). Human Sdc1 siRNA(target sequence GGAGGAATTCTATGCCTGA; SEQ ID NO. 2) and human Sdc4 siRNA(target sequence CAGGAATCTGATGACTTTGAG; SEQ ID NO. 3) were from Ambion.

Peptides comprising the SSTN_(EGFR) site in the Sdc4 extracellulardomain were synthesized by NeoBioLab, Inc. (Cambridge, Mass.). Theactivity of SSTN_(EGFR) was compared to other SSTN peptides beingdeveloped in the laboratory. These include SSTN_(HER2) (also obtainedfrom NeoBioLab, Inc.) and SSTN_(IGF1R), obtained from Genscript USA,Inc., (Piscataway, N.J.).

Plasmid Constructs.

The cDNAs of all syndecans were inserted into the pcDNA3 vector usingrestriction sites engineered into the 5′ ends of the primers used toamplify the syndecan fragments. Deletion of the C2 domain of mouse Sdc1and Sdc4 in pcDNA3 vector was made by inserting a stop codon before theEFYA sequence by using the Quikchange Site Directed Mutagenesis Kit(Stratagene, La Jolla, Calif.). The cDNA of human integrin β4 wereinserted into pcDNA vector. The cDNA of integrin β4 fragment encodingamino acids 1677-1752 was inserted into pTRC-His A vector; sitemutations and deletions were generated by using the Quikchange SiteDirected Mutagenesis Kit.

Cell Culture and Transfection.

Human HaCat keratinocytes, Head and Neck squamous carcinoma cell lines,and BT474 and SKBr3 mammary carcinoma cells were grown in DMEM,supplemented with 10% calf serum or 10% fetal bovine serum (HycloneLaboratories, Logan, Utah), 4 mM L-glutamine (Sigma), and 100 units/mlpenicillin and 100 g/ml streptomycin (Invitrogen) at 37° C. and 92.5%air, 7.5% CO2. MCF10A mammary epithelial cells were grown in DMEM F1250/50 plus 15 mM Hepes, L-glutamine, 5% horse serum, 10 μg/ml insulin,0.5 μg/ml hydrocortisone, and 0.02 μg/ml EGF. Cells were transfectedwith syndecan or integrin β4 constructs in pcDNA3 using LipofectaminePLUS (Invitrogen) and 10 μg of plasmid by following the manufacturer'sinstructions. Stable populations were selected in 1.0 mg/ml G418(Invitrogen).

siRNA Treatment and Flow Cytometry.

Oligos of siRNAs specific for human Sdc1 or Sdc4 were used as describedpreviously (Beauvais et al., 2004). To measure cell surface syndecanexpression, suspended cells were incubated for 1 h on ice with 1 μg ofprimary antibody per 3×10⁵ cells and then washed and counterstained withAlexa-488-conjugated secondary antibodies and scanned on a FACSCaliburbench top cytometer. Cell scatter and propidium iodide staining profileswere used to gate live, single-cell events for data analysis (Beauvaiset al., 2004; Beauvais et al., 2006).

Fusion Protein Expression and Purification.

6×His-β4CD1677-1752 fusion protein was expressed in E. coli by IPTGinduction and purified on Ni-NTA beads following cell lysis in 100 mMNaH2PO4, 10 mM Tris base and 8 M urea, (8.0). GST-S1CD or GST-S4CDfusion proteins and truncated versions were expressed in E. coli by IPTGinduced and purified on glutathione-sepharose beads following cell lysisin 150 mM NaCl, 20 mM sodium phosphate (pH 7.4) and 1% triton X-100.

Wound Healing Assay.

HaCat or MCF10A cells grown to confluence on 48 well-plates were starvedfor 6 hr by serum deprivation followed by introduction of a scratchwound in the monolayer using 200 μl pipette tip. To induce haptotacticmigration that depends on Sdc1, cells were cultured an additional 12 hrin DMEM containing mAb 3E1 (10 μg/ml), goat anti-mouse IgG (50 μg/ml)and 3 μM LPA. To cause EGF-stimulated chemotactic migration that dependson Sdc4, cells were stimulated with EGF (10 ng/ml). Images were acquiredusing a PlanApo 20 (0.75 numerical aperture) objective and aPhotometrics CoolSnap ES camera on a Nikon Eclipse TE2000U microscopysystem and wound closure quantified.

Immunoprecipitation.

Immunoprecipitations were carried out as described previously (Beauvaiset al., 2004; Beauvais et al., 2006). Cells were washed once withwashing buffer (50 mM Hepes, 50 mM NaCl and 10 mM EDTA, pH 7.4) andlysed for 20 min on ice in 1% Triton X-100 containing a 1:1000 dilutionof protease inhibitor mixture set III (Calbiochem) in washing buffer.Cell debris was removed by centrifugation at 20,000 g for 15 min at 4°C. Lysates or 6×His-tagged β4 cytoplasmic domain (amino acids 1677-1752)were incubated at 4° C. overnight with 100 μl Glutathione Sepharose 4Bbeads (50% in IP wash buffer), GST-S4CD or GST-S1CD in present or absentof different peptides. Samples were resolved by electrophoresis underreduced conditions on a 15% Laemmli gel, transferred to Immobilon P, andprobed with primary antibody followed by an alkalinephosphatase-conjugated secondary antibody. Visualization ofimmune-reactive bands was performed using ECF reagent (GE Healthcare)and scanned on a Typhoon Trio Variable Mode Imager (GE-Healthcare) inblue fluorescence.

Apoptosis Assay.

Cells (5×10⁴/well in a 24-well plate) were treated with GST fusionprotein (GST, GST-S1ED or GST-S4ED) or synstatin peptides ranging inconcentration from 1 to 30 μM. Cell death was observed by CellTilter-GLO Cell Viability Assay (Promega).

Example 2 Results

The inventor initially discovered using a yeast-two hybrid assay thatall four syndecans engage the cytoplasmic domain of the β4 integrin,relying in part on the conserved C2 domain at their extreme C-terminus(Wang et al., 2010) (see FIGS. 5A-B). Extending this to functionalstudies, the inventor found find that although all four syndecans engagethe integrin cytoplasmic domain, there is apparent specificity in whichsyndecan associates with the integrin when it is complexed withdifferent receptor tyrosine kinases. Focusing on syndecan-4 (Sdc4), hefound that Sdc4 associates with α6β4 integrin and EGFR.Immunoprecipitation of either Sdc4, the β4 integrin subunit (whichprecipitates the α6β4 integrin), or EGFR causes precipitation of EGFRfrom lysates of head and neck squamous carcinoma (HNSCC) cells (FIG. 2)and from breast epithelial cells, including the normal MCF10A cells orthe breast carcinoma BT474 (FIG. 3). The inventor found that this isspecific for Sdc4, as immunoprecipitation of Sdc1 (as a comparison)co-precipitates the integrin (not shown), but fails to co-precipitateEGFR (shown for the breast epithelial cells in FIG. 3).

It is known that keratinocyte migration depends on the α6β4 and mimics anormal skin wound healing mechanism that occurs in response to EGF. Theα6β4 and α3β1 integrins collaborate to deposit laminin (LN332), which isthe ligand recognized by these two integrins, and then use the integrinsfor the signaling mechanism necessary for migration on this substratum(Russell et al., 2003; Sehgal et al., 2006). Testing the migration ofhuman HaCat keratinocytes in a scratch wound assay in which EGFstimulates wound closure, the inventor found that blocking either ofthese two integrins, or blocking their binding site on LN332, disruptswound closure, as expected (FIG. 4A). The inventor also found thatmigration is not inhibited by 10 μM tyrphostin AG825, which blocks thekinase activity of the EGFR family member HER2 but does not block EGFR;in contrast, the EGFR-specific kinase inhibitor Iressa (getintinib) usedat 1 μM, which fails to block HER2 but inhibits EGFR, does block woundclosure, confirming that the signaling mechanism leading to cellmigration depends on the EGFR (FIG. 4A). Next, the inventor silenced theexpression of the two main syndecans expressed on the HaCat cells—Sdc1and Sdc4. He found that silencing human Sdc1 expression has only aminimal effect on cell migration, an effect that can be rescued byexpression of mouse Sdc1 (which is not affected by the siRNA used tosilence the human form), but cannot be rescued by Sdc1 lacking its C2region (mSdc1ΔC2) necessary for engaging the α6β4 integrin cytoplasmicdomain. This cytoplasmic domain engagement by the syndecan is necessaryfor phosphorylation of the integrin by the HER2 kinase (Wang et al.,2010), suggesting that Sdc1 engagement with the integrin may have somerole, although a minor one, in the migration mechanism. In contrast,silencing Sdc4 expression reduces migration to that of non-stimulatedcells (e.g., no EGF addition). This migration is rescued by expressionof mouse Sdc4, but cannot be rescued by Sdc4^(ΔC2) that fails to engagethe β4 integrin cytoplasmic domain (FIG. 4B). This implicates Sdc4 andits interaction with the integrin during EGF stimulated migration,likely reflecting the association observed via Immunoprecipitation inFIGS. 2 and 3.

Further comparing the integrin association with either Sdc1 or Sdc4, theinventor found that the syndecans rely on distinct and highly specificcytoplasmic and extracellular motifs to assemble these receptorcomplexes and that these specific interactions dictate whether themechanism will rely on Sdc1 and HER2 kinase to cause haptotacticmigration, or Sdc4 and EGFR kinase for EGF-stimulated chemotaxis.Binding motifs near the C-terminus of both Sdc1 and Sdc4 engage bindingsites near the C-terminus of the β4 integrin, but these binding sitesare distinct for the two syndecans (FIGS. 5A-B). At least one amino acidwithin the EFYA sequence (the C2 region) that both syndecans share attheir extreme C-terminus seems to be required (explaining why the ΔC2mutants fail to engage the integrin), but additional amino acids in theV region that is not shared between the two receptors also have a role.In the β4 integrin, the binding site for both syndecans is within thelast 24 amino acids at its C-terminus, as deletion of this regionabolishes binding for both syndecans. The inventor found that E1729 inthe β4 cytoplasmic domain is highly specific for Sdc4, as its mutationto alanine (β4^(E1729A) mutant) reduces its affinity for Sdc4 withoutaffecting its affinity for Sdc1. Conversely, R1733 is highly specificfor Sdc1, as its mutation to alanine (β4^(R1733A) mutant) reduces itsaffinity for Sdc1 but not Sdc4. These two mutants act as dominantnegative mutants when expressed in cells; that is, the E1729A mutantcompetes for the Sdc4-coupled mechanism and blocks it, whereas theR1733A mutant competes for and blocks the Sdc1-coupled mechanism. Whenexpressed in HaCat or MCF10A cells that appear to rely on Sdc4 forEGF-stimulated chemotaxis (based on siRNA silencing of Sdc4), theinventor found that the β4^(E1729A) mutant that disrupts theSdc4-specific signaling mechanism blocks the migratory response of thecells to EGF; In contrast, the Sdc1-specific β4^(R1733A) mutant does notblock the response, providing additional support for the hypothesis thatSdc4 and its coupling to the α6β4 integrin is necessary for thisEGF-mediated signaling (FIG. 7).

The inventor's prior work has shown that Sdc1 contains a site in itsextracellular domain to capture the αvβ3 or αvβ5 integrin and theinsulin-like growth factor-1 receptor (IGF1-R) (Beauvais et al., 2009;Beauvais & Rapraeger, 2010). This capture is necessary for thesereceptors to cause carcinoma cell migration and for endothelial cells toundergo angiogenesis. This work acts as a model for the Sdc4-EGFRsignaling mechanism and suggests that Sdc4 may also rely on a specificsite in its extracellular domain to capture the α6β4 integrin and/orEGFR necessary for EGF-stimulated chemotaxis, in addition to itsspecific interaction with the cytoplasmic domain of the α6β4 integrin.To test this, the inventor swapped the extracellular domain of Sdc1,which does not appear to participate in EGF chemotaxis, with that ofSdc4, and then tested the ability of these chimeras to co-precipitatewith EGFR from HaCat keratinocytes. He found that Sdc1 bearing theectodomain of Sdc4 now assembles with EGFR (FIG. 6A), whereas Sdc4displaying the Sdc1 ectodomain has lost this ability. Next,demonstrating that this capture is dependent solely on the syndecanectodomain, the inventor found that beads coated with recombinant Sdc4ectodomain (GST-S4ED) captures EGFR from HaCat cell lysates (FIG. 6B).Re-probing the blot shows that α6β4 integrin is also captured (data notshown), suggesting it is the α6β4 integrin/EGFR together that arecaptured via an interaction site in the Sdc4 ectodomain. In contrast,GST-S1ED fails to capture EGFR, but does capture HER2, coinciding withthe role of Sdc1 in α6β4/HER2-stimulated haptotaxis (Wang et al., 2010).

These findings suggest that the recombinant Sdc4 extracellular domaincontains a specific interaction site necessary for capture of EGFRand/or the α6β4 integrin, and that a peptide representing this sitemight serve to disrupt the α6β4/EGFR signaling mechanism. Indeed, theinventor found that competition with the entire recombinant S4EDdisrupts the EGF-stimulated chemotaxis of MCF10A human mammaryepithelial cells (FIG. 7) or HaCat keratinocytes (data not shown) withan IC₅₀ of 0.1-0.3 μM (see summary in FIGS. 8A-C). Both mouse and humanproteins compete with the same affinity. In contrast, the ectodomain ofSdc1 (S1ED) fails to display any inhibitory effect. The inventor nextused this assay to further truncate the recombinant S4ED protein to findthe smallest peptide that retains full inhibitory activity and whichwould be a new synstatin for potential therapeutic use (e.g.,SSTN_(EGFR)). The mature human Sdc4 ectodomain (e.g., lacking its signalpeptide) is 127 amino acids in length. The inventor found that anytruncated version of this protein that retains the sequence betweenamino acids 87-131 of human Sdc4 retains full competitive activity (FIG.8A), suggesting that the active motif is contained within this 45 aminoacid sequence. Indeed, a peptide containing these amino acids, which iscalled SSTN_(EGFR), blocks EGF-stimulated MCF10A cell migration with anIC₅₀ of 0.1-0.3 mM (FIG. 7). There are several regions of conservationwithin this sequence (FIG. 8B), especially an NxIP motif at theN-terminus of the competitive peptide, an internal NEV motif, and anSNKVSM motif at the C-terminal end. Mutation of NxIP to NxAP reduces thecompetitive activity 10-fold, either in the recombinant S4ED fusionprotein (FIG. 8A) or in the synthetic peptide comprising amino acids87-131 (FIG. 8C). Mutation of the NEV motif to AAA appears to havelittle if any effect (FIG. 8A). But truncation of the SNKVSM motifcauses over a 100-fold loss of activity (FIG. 8C). Thus, the 87-131sequence comprises a nearly minimal peptide that retains fullcompetitive activity. In addition to the NxIP and SNKVSM (SEQ ID NO: 6)motifs that define its N- and C-terminal ends, an internal VPTEPKxLEE(SEQ ID NO: 7) motif conserved in mammals is also important, as a10-fold loss of activity is observed if this sequence is mutated toAAAAAAAAAA (SEQ ID NO: 8) in the synthetic SSTN_(EGFR) peptide (FIG.8C).

Signaling from growth factor receptors and integrins is often criticalnot only for cell migration and tumor cell invasion, but also for cellproliferation and survival. Thus, the inventor has tested the potentialrole of the Sdc4-coupled α6β4/EGFR mechanism on the growth and survivalof either normal epithelial or carcinoma cells using the SSTN_(EGFR)peptide. The inventor tested the peptide on the HaCat keratinocytes thathe has used for migration studies, an example of a normal stratifiedepithelium, and against the MCF10A cells that he has also used inmigration studies, an example of a normal human breast epithelium. Hefound that although SSTN_(EGFR) blocks EGF-stimulated chemotaxis ofthese normal cells, it has no effect on their growth or survival whenused at concentrations as high as 30 μM (FIGS. 9A-F). The inventor alsotested the peptide in combination with other SSTN peptides that hedeveloped: SSTN_(IGF1R) that targets a Sdc1-αvβ3 integrin-IGF1R receptorcomplex, and SSTN_(HER2), that targets a Sdc1-α6β4 integrin-HER2 kinasecomplex. None of these SSTNs or SSTN combinations affect the normalepithelial cells. In contrast, however, SSTN_(EGFR) and the other SSTNsdo target growth/survival mechanisms in the carcinoma cells. Thisincludes the UM-SCC47 and SCC25 squamous carcinoma of the humantongue—tumors that are derived from stratified epithelia as are thenormal HaCat keratinocytes—and MDA-MB-468 and SKBr3 human breastcarcinoma cells, representative of tumors arising from breastepithelium. These experiments represent relatively short treatment times(4 or 7 days). Nonetheless, cell death is observed in those cases wherepeptide combinations are used (shown by a reduction in cell number tofewer cells than the number present at the start of the assay). Thus,remarkably, tumor cells but not normal epithelial cells rely on theSdc4-α6β4-EGFR complex for their growth and survival, and SSTN_(EGFR)effectively blocks this dependence, especially when used in combinationwith other SSTN peptides.

The inventor has tested the entire recombinant S4ED protein (as aGST-S4ED fusion protein) as an inhibitor against one of these carcinomacells (the MDA-MB-468 cells) during tumor growth in immunocompromisednude (nu/nu) mice. Note that at 400 μM in the pump (estimated 2-3 μM inthe blood), the GST-S4ED begins to disrupts the growth of MDA-MB-468breast carcinoma after 2 weeks, with the tumor growth leveling off afterthat time, while the untreated tumors or tumors treated with GST alonecontinue to grow (FIG. 10).

Example 3 Discussion

Epithelial cells rely on the α6β4 integrin to form hemidesmosomes,anchoring the epithelial layer to the ECM and helping it to resistfrictional forces. However, during wound healing or normal epithelia, orin transformed carcinomas overexpressing the EGFR, the hemidesmosomesbreak down in response to EGFR signaling and the free integrinassociates with the EGFR. EGFR causes phosphorylation of the β4 subunitin its signaling domain, leading to cell proliferation, survival (if acarcinoma) and invasion. The current work shows that this mechanismrelies on association of the integrin with EGFR and Sdc4. Sdc4 engagesthe cytoplasmic domain of the integrin, ostensibly bringing it to themembrane where it becomes phosphorylated. But a site in theextracellular domain of Sdc4 is also essential and appears responsiblefor capturing α6β4 integrin and EGFR as a signaling complex. Competitionwith either full-length Sdc4 ectodomain expressed as a recombinantprotein, or competition with a peptide consisting of amino acids 87-131in Sdc4 (human sequence) serves to block the EGF stimulated migration ofepithelia, and disrupts the proliferation and survival of tumor cellsthat depend on this mechanism. The inventor proposes that this sequence,called synstatin-EGFR or SSTN_(EGFR), is a new anti-cancer therapeutic.It acts on breast carcinoma cells, as well as on squamous cellcarcinoma—two examples of carcinoma that rely on this signalingmechanism, apparently by competing for the assembly of α6β4 and EGFRwith Sdc4. Blockade of this assembly, at a minimum, prevents activationof the α6β4 integrin and may have additional effects on activation ofthe EGFR as well. Importantly, although blockade of assembly disruptsthe migration of normal cells, it does not lead to cell death as it doesfor tumor cells.

The region of Sdc4 that contains the active motif for SSTN_(EGFR) haspreviously been recognized to bind cell surface receptors on other typesof cells (McFall et al., 1997; 1998; Whiteford and Couchman, 2006).demonstrated that recombinant Sdc4 ectodomain used as an attachmentsubstratum captured fibroblasts and endothelial cells, indicating thatreceptors on those cells interact with this protein. Expressingtruncated versions of the protein, she identified the active region asamino acids 78-131 (actually referred to as 56-109 in her work (McFallet al., 1997; 1998) after subtraction of the signal peptide sequence),similar to the present work. However, there are several importantdifferences between the reports of McFall et al. and the present work.First, McFall envisioned that shed Sdc4 might act as a matrix-boundligand and thus provide a site for cell-matrix adhesion. The presentwork proposes that Sdc4 acts as an organizer to cause assembly ofreceptors into signaling complexes in cis (e.g., on the same cell).McFall's finding has been followed up by work from Whiteford et al.(2006), again describing the protein as an adhesion ligand.

Secondly, McFall described the activity on fibroblasts and endothelialcells; this is in contrast to the present work as the α6β4 integrin—thetarget of the present work—is not expressed on fibroblasts, nor is itexpressed on endothelial cells when they are grown in culture (Homan etal. 1998). Whiteford et al. (2006) also described the protein as aligand for fibroblastic, endothelial and lymphoblastic cells, butreported that it fails to act as an adhesion ligand for epithelialcells, which express the α6β4 integrin. Thus, it seems that the assaydevised by McFall et al. and Whiteford et al. was reporting on adhesionvia a different receptor—one found on fibroblastic and not epithelialcells, whereas this report is for receptor(s) found on epithelia.

Last, McFall also tested truncation mutants of the active sequence fortheir ability to mimic binding to the surface of fibroblasts, findingthat S4ED78-118 was completely inactive as a competitor for celladhesion to full-length S4ED. In contrast, S4ED 98-131 could compete,but was 100-fold less active that S4ED78-131. This contrasts with thepresent findings (FIGS. 8A-C) in which S4ED 78-118 and S4ED98-131equally exhibit ca. 10% competitive activity, suggesting that they aretargeting a different site or set of receptors than McFall et al.

The inventor's conclusions from these findings are that the S4ED87-131motif may be a structural unit that presents different binding motifsfor capture of different receptors. Thus, he proposes that a specificset of amino acids within this region binds to (and competes for) a setof receptors on carcinoma cells that is distinct from the receptor(s)studied by McFall et al. and Whiteford et al. He envisions that thisspecific inhibitor will be highly specific for the Sdc4/α6β4/EGFRreceptor complex and its role in promoting epithelial cancers andangiogenesis.

The α6β4 integrin appears to be expressed with HER2 and EGFR in thevasculature and lymphatics of tumors (Nikolopoulos et al., 2004; Amin etal., 2006; Bohling et al., 1996; Bruns et al., 2000; Kedar et al., 2002;Huang et al., 2002), potentially implicating the Sdc4-coupled mechanismsin angiogenesis. The role of the α6β4 integrin in vascular endothelialcells is not well appreciated, as vascular endothelial cells rapidlylose expression of α6β4 integrin when placed into culture, making itdifficult to study (Homan et al., 1998). When expressed artificially insuch cells, it is found that they rely on α6β4 integrin to activate Erkand NFκB signaling pathways and migrate to close scratch wounds whenplated on LN332 (Nikolopoulos et al., 2004), similar to its role innormal epithelial cells. Work from mouse models of tumorigenesis in vivoare more clear, demonstrating that not only is the integrin expressed intumor vasculature, but HER2 and EGFR are also expressed, especially inendothelial cells lining blood vessels surrounding tumors (Amin et al.,2006; Bruns et al., 2000; Kedar et al., 2002), and that HER2 and EGFRcouple with the α6β4 integrin during tumor-induced angiogenesis(Nikolopoulos et al., 2004). Both Sdc1 and Sdc4 are expressed in thevasculature as well, including tumor vasculature (Beauvais et al., 2009;Echtermeyer et al., 2001; Partovian et al., 2008; Tkachenko et al.,2004). Thus, although the inventor has not yet studied its effects ontumor-induced angiogenesis, it is highly plausible that SSTN_(EGFR)targets tumor angiogenesis as well as the growth, survival and invasionof the tumor cells themselves.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

VII. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

-   U.S. Pat. No. 5,440,013-   U.S. Pat. No. 5,446,128-   U.S. Pat. No. 5,475,085-   U.S. Pat. No. 5,597,457-   U.S. Pat. No. 5,618,914-   U.S. Pat. No. 5,670,155-   U.S. Pat. No. 5,672,681-   U.S. Pat. No. 5,674,976-   U.S. Pat. No. 5,710,245-   U.S. Pat. No. 5,790,421-   U.S. Pat. No. 5,840,833-   U.S. Pat. No. 5,859,184-   U.S. Pat. No. 5,889,155-   U.S. Pat. No. 5,929,237-   U.S. Pat. No. 6,093,573-   U.S. Pat. No. 6,261,569-   U.S. Pat. No. 7,183,059-   U.S. Pat. No. 7,192,713-   U.S. Patent Appln. 2005/0015232-   Agazie and Hayman, Mol. Cell. Biol., 23(21):7875-7886, 2003.-   Amano et al., J. Biol. Chem., 275(30):22728-22735, 2000.-   Amin et al., Cancer Res., 66(4):2173-2180, 2006.-   Baciu and Goetinck, Mol. Biol. Cell, 6:1503-1513, 1995.-   Beauvais et al., J. Cell Biol., 167(1):171-181, 2004.-   Beauvais et al., J. Exp. Med., 206(3):691-705, 2009.-   Beauvais and Rapraeger, J Cell Sci, 123(21): p. 3796-807, 2010.-   Bernfield et al., Annu. Rev. Biochem., 68:729-777, 1999.-   Bernfield et al., Annu. Rev. Cell Biol., 8:365-393, 1992.-   Bertotti et al., Cancer Res., 65(23):10674-10679, 2005.-   Bertotti et al., J. Cell Biol., 175(6):993-1003, 2006.-   Bodanszky et al., J. Antibiot., 29(5):549-53, 1976.-   Bohling et al., J Neuropathol Exp Neurol, 1996. 55(5): p. 522-7,    1996.-   Bon et al., Breast Cancer Res, 9(1):203, 2007.-   Boudreau et al., J. Cell Biol., 139(1):257-264, 1997.-   Brooks et al., Cell, 79:1157-1164, 1994.-   Bruns et al., Cancer Res., 60(11):2926-2935, 2000.-   Carey et al., Exp. Cell Res., 214:12-21, 1994a.-   Carey et al., J. Cell Biol., 124:161-170, 1994b.-   Carey et al., Otolaryngol. Head Neck Surg., 96(3):221-230, 1987.-   Carulli et al., J. Biol. Chem., 287(15):12204-12216, 2012.-   Carvalho et al., Clinics (Sao Paulo), 65(10):1033-1036, 2010.-   Cassell and Grandis, Expert Opin. Investig. Drugs, 19(6):709-722,    2010.-   Chang and Califano, J. Surg. Oncol., 97(8):640-643, 2008.-   Choi and Chen, Cancer, 104(6):1113-1128, 2005.-   Chung et al., Cancer Cell, 5(5):489-500, 2004.-   Cohen et al., J. Med. Chem., 33:883-894, 1990.-   Colorado et al., Cancer Res., 60(9):2520-2526, 2000.-   Couchman et al., Int. Rev. Cytol., 207:113-150, 2001.-   Dajee et al., Nature, 421(6923):639-643, 2003.-   Dans et al., J. Biol. Chem., 276(2):1494-1502, 2001.-   Datta et al., Genes Dev., 13(22):2905-2927, 1999.-   David et al., J. Cell Biol., 118(4):961-969, 1992.-   Dent et al., Clin. Cancer Res., 13(15 Pt 1):4429-4434, 2007.-   Dent et al., Mol. Biol. Cell, 10(8):2493-2506, 1999.-   Diaz et al., Mod. Pathol., 18(9):1165-1175, 2005.-   Dutta and Shaw, Cancer Res., 68(21):8779-8787, 2008.-   Echtermeyer et al., J Clin Invest, 107(2): p. R9-R14, 2001.-   Elenius et al., J. Cell Biol., 114(3):585-595, 1991.-   Falcioni et al., Cancer Res., 46(11):5772-5778, 1986.-   Falcioni et al., Exp. Cell Res., 236(1):76-85, 1997.-   Folgiero et al., PLoS One, 3(2):e1592, 2008.-   Friedlander et al., Science, 270:1500-1502, 1995.-   Friedrichs et al., Cancer Res., 55(4):901-906, 1995.-   Gallo et al., J. Invest. Dermatol., 107(5):676-683, 1996.-   Gambaletta et al., J. Biol. Chem., 275(14):10604-10610, 2000.-   Giancotti, Trends Pharmacol. Sci., 28(10):506-511, 2007.-   Ginos et al., Cancer Res., 64(1):55-63, 2004.-   Goldfinger et al., J. Cell Biol., 141(1):255-265, 1998.-   Goldfinger et al., J. Cell Sci., 112(Pt 16):2615-2629, 1999.-   Gotte et al., Invest. Ophthal. Visual Sci., 43(4):1135-1141, 2002.-   Grandis et al., Oncogene, 15(4):409-416, 1997.-   Granes et al., Exp. Cell Res., 248:439-456, 1999.-   Gronenborn et al., Anal. Chem., 62(1):2-15, 1990.-   Guo et al., Cell, 126(3):489-502, 2006.-   Haffty et al., J. Clin. Oncol., 24(36):5652-5657, 2006.-   Hansen et al., J. Cell Biol., 126:811-819, 1994.-   Haupt et al., Arch. Pathol. Lab. Med., 134(1):130-133, 2010.-   Herschkowitz et al., Genome Biol., 8(5):R76, 2007.-   Homan et al., J Cell Sci, 111(18): p. 2717-28, 1998.-   Hopkinson and Jones, Mol. Biol. Cell, 11(1):277-286, 2000.-   Huang et al., Mol. Cancer Ther., 1(7):507-514, 2002.-   Hynes and Lane, Nat. Rev. Cancer, 5(5): 341-354, 2005.-   Hynes and MacDonald, Curr. Opin. Cell Biol., 21(2):177-184, 2009.-   Iba et al., J. Cell Biol., 149:1143-1156, 2000.-   Izzard et al., Exp. Cell Res., 165:320-336, 1986.-   Jackson, Seminars in Oncology, 24:L164-172, 1997.-   Johnson et al., In: Biotechnology And Pharmacy, Pezzuto et al.    (Eds.), Chapman and Hall, NY, 1993.-   Jones et al., J. Med. Chem., 39:904-917, 1996.-   Kamphaus et al., J. Biol. Chem., 275(2):1209-1215, 2000.-   Kedar et al., Clin. Cancer Res., 8(11):3592-3600, 2002.-   Khan et al., J. Biol. Chem., 263:11314-113148, 1988.-   Kimmel and Carey, Cancer Res., 46(7):3614-3623, 1986.-   Klass et al., J. Cell Sci., 113:493-506, 2000.-   Lebakken, and Rapraeger, J. Cell Biol., 132:1209-1221, 1996.-   Liu et al., J. Biol. Chem., 273:22825-22832, 1998.-   Livasy et al., Mod. Pathol., 19(2):264-271, 2006.-   Lu et al., Clin. Cancer Res., 14(4):1050-1058, 2008.-   Maeshima et al., J. Biol. Chem., 275(28):21340-21348, 2000.-   Mainiero et al., EMBO J., 16(9): 2365-2375, 1997.-   Mainiero et al., J. Cell Biol., 134(1):241-253, 1996.-   Marinkovich et al., J. Biol. Chem., 267(25):17900-17906, 1992.-   Mariotti et al., J. Cell Biol., 155(3):447-458, 2001.-   Matsui et al., J. Biol. Chem., 270(40):23496-23503, 1995.-   McFall and Rapraeger, J. Biol. Chem., 272:12901-12904, 1997.-   McFall and Rapraeger, J. Biol. Chem., 273:28270-28276, 1998.-   McPherson, J. Biol. Chem., 251:6300-6306, 1976.-   Merdek et al., J. Biol. Chem., 282(41):30322-30330, 2007.-   Merrifield, J. Am. Chem. Soc., 85:2149-2154, 1963.-   Mertens et al., J. Biol. Chem., 267(28):20435-20443, 1992.-   Miranti and Brugge, Nat. Cell Biol., 4:E83-90, 2002.-   Myers et al., Am. J. Pathology, 161(6): 2099-2109, 2002.-   Myers et al., J. Cell Biol., 148(2): 343-351, 2000.-   Navia et al., Curr. Opin. Struct. Biol., 2:202-210, 1992.-   Nievers et al., Matrix Biol., 18(1):5-17, 1999.-   Nikolopoulos et al., Cancer Cell, 6(5):471-483, 2004.-   Oh et al., J. Biol. Chem., 272:8133-8136, 1997a.-   Oh et al., J. Biol. Chem., 272:11805-11811, 1997b.-   Oh et al., J. Biol. Chem., 273:10624-10629, 1998.-   Ohtake et al., Br. J. Cancer, 81:393-403, 1999.-   O'Reilly et al., Cell, 79(2):315-328, 1994.-   O'Reilly et al., Cell, 88(2):277-285, 1997.-   Partovian et al., Mol Cell, 32(1): p. 140-9, 2008.-   Patarroyo et al., Semin. Cancer Biol., 12(3):197-207, 2002.-   PCT Appln. PCT/US00/03745-   PCT Appln.PCT/US00/14667-   PCT Appln.PCT/US99/11913-   PCT Appln.PCT/US99/18441-   Peptide Synthesis, 1985-   Perez-Ordonez et al., J. Clin. Pathol., 59(5):445-453, 2006.-   Perou et al., Nature, 406(6797):747-752, 2000.-   Protective Groups in Organic Chemistry, 1973-   Protein NMR Spectroscopy, Principles and Practice, J. Cavanagh et    al., Academic Press, San Diego, 1996.-   Rabinovitz et al., Mol. Cell Biol., 24(10):4351-4360, 2004.-   Rapraeger and Ott, Curr. Opin. Cell Biol., 10(5):620-628, 1998.-   Rapraeger, J. Cell Biol., 149:995-998, 2000.-   Remington's Pharmaceutical Sciences, 15^(th) Ed., 1035-1038 and    1570-1580, 1990.-   Remington's Pharmaceutical Sciences, 15^(th) Ed., 3:624-652, 1990.-   Roskelley et al., Curr. Opin. Cell Biol., 7:736-747, 1995.-   Russell et al., J. Cell Sci., 116(Pt 17):3543-3556, 2003.-   Santoro et al., Dev. Cell., 5(2):257-271, 2003.-   Saoncella et al., Proc. Natl. Acad. Sci. USA, 96:2805-2810, 1999.-   Scaltriti and Baselga, Clin. Cancer Res., 12(18):5268-5272, 2006.-   Schafineister et al., J. Amer. Chem. Soc., 122(24) 5891-5892, 2000.-   Schmidt-Ullrich et al., Int. J. Radiat. Oncol. Biol. Phys.,    29(4):813-819., 1994-   Sehgal et al., J. Biol. Chem., 281(46):35487-35498, 2006.-   Shaw et al., Cell, 91(7):949-960, 1997.-   Shaw, Mol. Cell Biol., 21(15):5082-5093, 2001.-   Singer et al., J. Cell Biol., 104:573-584, 1987.-   Solid Phase Peptide Synthelia, 1984-   Streeter and Rees, J. Cell Biol., 105:507-515, 1987.-   Teng et al., Breast Cancer Res., 13(2):R35, 2011.-   Tkachenko et al., J Cell Sci, 117(15): p. 3189-99, 2004.-   Tran et al., Cancer Res., 68(8):2885-2894, 2008.-   Trusolino et al., Cell, 107(5):643-654, 2001.-   Tsuruta et al., Curr. Med. Chem., 15(20):1968-1975, 2008.-   Van Waes et al., Cancer Res., 51(9):2395-2402, 1991.-   Wang et al., J. Biol. Chem., 285:13569-13579, 2010.-   Weber, Advances Protein Chem., 41:1-36, 1991.-   Whiteford and Couchman, J Biol Chem, 281(43): p. 32156-63, 2006.-   Wider, BioTechniques, 29:1278-1294, 2000.-   Wilhelmsen et al., Mol. Biol. Cell, 18(9):3512-3522, 2007.-   Wilhelmsen et al., Mol. Cell Biol., 26(8):2877-2886, 2006.-   Wolf et al., J. Natl. Cancer Inst., 82(19):1566-1572., 1990-   Woods and Couchman, Curr. Opin. Cell Biol., 13:578-583, 2001.-   Woods and Couchman, Mol. Biol. Cell, 5:183-192, 1994.-   Woods et al., Embo J., 5:665-670, 1986.-   Yamashita et al., J. Immunol., 162:5940-5948, 1999.-   Yang et al., Mol. Cell. Biol., 30(22):5306-5317, 2010.-   Zimmermann et al., Radiat. Oncol., 1:11, 2006.

1. An isolated and purified peptide segment consisting of between 25 and100 amino acid residues and comprising about 45 residues of SEQ ID NO:1including residues 87-131 (SEQ ID NO: 4).
 2. The isolated and purifiedpeptide of claim 1, wherein said peptide is 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acidresidues in length.
 3. The isolated and purified peptide of claim 1,wherein said peptide is between 45 and 54 amino acid residues in length.4. The isolated and purified peptide of claim 1, wherein said peptide isbetween 45 and 65 amino acid residues in length.
 5. The isolated andpurified peptide of claim 1, wherein said peptide is between 45 and 75amino acid residues in length.
 6. The isolated and purified peptide ofclaim 1, wherein said peptide consists essentially of residues 87-131(SEQ ID NO: 4).
 7. The isolated and purified peptide of claim 1, whereinsaid peptide consists of residues 87-131 (SEQ ID NO: 4).
 8. The isolatedand purified peptide of claim 1, wherein said peptide consists ofessentially of residues 78-131 (SEQ ID NO: 5).
 9. The isolated andpurified peptide of claim 1, wherein said peptide consists of residues78-131 (SEQ ID NO: 5).
 10. The isolated and purified peptide of claim 1,wherein said peptide comprises all D amino acid, all L amino acids, or amixture of D and L amino acids.
 11. A nucleic acid encoding a peptidesegment consisting of between 45 and 100 amino acid residues andcomprising about 45 residues of SEQ ID NO:1 including residues 87-131(SEQ ID NO: 4). 12-17. (canceled)
 18. The nucleic acid of claim 11,wherein said nucleic acid is in operable connection to a promoter. 19.The nucleic acid of claim 11, wherein said nucleic acid is located in areplicable vector.
 20. The nucleic acid of claim 19, wherein said vectoris a viral vector.
 21. A method of inhibiting α6β₄ integrin interactionwith EGFR comprising contacting a EGFR molecule with a peptide segmentconsisting of between 45 and 100 amino acid residues and comprisingabout 45 residues of SEQ ID NO:1 including residues 87-131 (SEQ ID NO:4). 22-29. (canceled)
 30. The method of claim 21, wherein said α6β4integrin is located on the surface of a cell.
 31. The method of claim30, wherein said cell is a cancer cell. 32-45. (canceled)
 46. A methodof treating a subject with a cancer, cancer cells of which express α6β₄integrin and EGFR, comprising contacting said cells with a peptidesegment consisting of between 45 and 100 amino acid residues andcomprising about 45 residues of SEQ ID NO:1 including residues 87-131(SEQ ID NO: 4). 47-55. (canceled)
 56. The method of claim 46, whereinsaid cancer is a carcinoma, a myeloma, a melanoma or a glioma. 57-60.(canceled)
 61. A method of inhibiting scarring in a subject tocomprising administering to said subject a peptide segment consisting ofbetween 45 and 100 amino acid residues and comprising about 45 residuesof SEQ ID NO:1 including residues 87-131 (SEQ ID NO: 4). 62-69.(canceled)
 70. A method of inhibiting pathologic neovascularizationcomprising administering to said subject a peptide segment consisting ofbetween 45 and 100 amino acid residues and comprising about 45 residuesof SEQ ID NO:1 including residues 87-131 (SEQ ID NO: 4). 71-79.(canceled)